Melanocortin Receptor-Specific Peptide with C-Terminal Naphthylalanine

ABSTRACT

Melanocortin receptor-specific peptides with a C-terminal naphthylalanine of the formula: 
     
       
         
         
             
             
         
       
     
     where R 4 , R 7 , R 9 , R 11 , R 18 , R 20 , R 21a , R 21b , R 21c  and R 22  are as defined in the claims, compositions and formulations including the peptides of the foregoing formula or salts thereof, and methods of preventing, ameliorating or treating melanocortin receptor-mediated or responsive diseases, indications, conditions and syndromes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/268,761, entitled “Melanocortin-1 Receptor-Specific CyclicHexapeptides”, filed on Sep. 19, 2016, which in turn is a continuationapplication of U.S. Ser. No. 14/514,472, entitled “Melanocortin-1Receptor-Specific Cyclic Peptides,” filed on Oct. 15, 2014, now U.S.Pat. No. 9,447,148, issued Sep. 20, 2016, which in turn is a divisionalapplication of U.S. Ser. No. 13/910,422, entitled “Melanocortin-1Receptor-Specific Cyclic Peptides,” filed on Jun. 5, 2013, now U.S. Pat.No. 8,877,890, issued Nov. 4, 2014, which in turn is a continuationapplication of U.S. Ser. No. 13/472,914, entitled “Melanocortin-1Receptor-Specific Cyclic Peptides,” filed on May 16, 2012, now U.S. Pat.No. 8,492,517, issued Jul. 23, 2013. U.S. Ser. No. 13/472,914 is acontinuation of International Application No. PCT/US2010/057699,published as International Publication No. WO 2011/063366, entitled“Melanocortin-1 Receptor-Specific Cyclic Peptides”, filed on Nov. 23,2010, which in turn claimed priority to and the benefit of the filing ofU.S. Provisional Patent Application Ser. No. 61/263,490 entitled“Melanocortin-1 Receptor-Specific Cyclic Peptides”, filed on Nov. 23,2009. The specification and claims of each of the foregoing applicationsare incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention (Technical Field)

The present invention relates to melanocortin receptor-specific cyclicpeptides, particularly cyclic peptides selective and specific for themelanocortin-1 receptor, which may be used in the treatment ofmelanocortin-1 receptor-mediated or responsive diseases, indications,conditions and syndromes.

Description of Related Art

The following discussion refers to a number of publications by author(s)and year of publication, and that due to recent publication datescertain publications are not to be considered as prior art vis-a-vis thepresent invention. Discussion of such publications herein is given formore complete background and is not to be construed as an admission thatsuch publications are prior art for patentability determinationpurposes.

A family of melanocortin receptor types and subtypes has beenidentified. Receptor types include melanocortin-1 (MC-1) receptor(MCR-1), commonly known to be expressed in normal human melanocytes andon melanoma cells, but which is also reported to be expressed in variousother cells, including those involved in immune responses, such asmonocytes, neutrophils, lymphocytes, dendritic cells, natural killer(NK) cells and endothelial cells. See generally, Kang, L., et al., “Aselective small molecule agonist of melanocortin-1 receptor inhibitslipopolysaccharide-induced cytokine accumulation and leukocyteinfiltration in mice,” J. Leuk. Biol. 80:897-904 (2006), and referencescited therein. A variety of human MCR-1 subtypes and variants are known,including those disclosed in U.S. Pat. Nos. 6,693,184 and 7,115,393. Inaddition to MCR-1, other melanocortin receptor types includemelanocortin-2 receptor (MCR-2) for ACTH (adrenocorticotropin),expressed in cells of the adrenal gland, melanocortin-3 andmelanocortin-4 (MC-4) receptors (MCR-3 and MCR-4), expressed primarilyin cells in the hypothalamus, mid-brain and brainstem, andmelanocortin-5 receptor (MCR-5), expressed in a wide distribution ofperipheral tissues.

The primary endogenous melanocortin agonist is the cyclicα-melanocyte-stimulating hormone (“α-MSH”) peptide. Melanocortinreceptor-specific peptides generally contain the central tetrapeptidesequence of native α-MSH, His⁶-Phe⁷-Arg⁸-Trp⁹ (SEQ ID NO:1), or amimetic or variation thereof, including various substitutions at one ormore positions (see, e.g., Hruby, V. J., et al., “Alpha-Melanotropin:the minimal active sequence in the frog skin bioassay,” J. Med. Chem.,30:2126-2130 (1987); Castrucci, A. M. L., et al., “Alpha-melanotropin:the minimal active sequence in the lizard skin bioassay,” Gen. Comp.Endocrinol., 73:157-163 (1989); Haskell-Luevano, C., et al., “Discoveryof prototype peptidomimetic agonists at the human melanocortin receptorsMC1R and MC4R,” J. Med. Chem., 40:2133-2139 (1997); Holder, J. R., etal., “Structure-activity relationships of the melanocortin tetrapeptideAc-His-DPhe-Arg-Trp-NH₂. 1. Modifications at the His position,” J. Med.Chem., 45:2801-2810 (2002); Abdel-Malek, Z. A., et al., “Melanomaprevention strategy based on using tetrapeptide α-MSH analogs thatprotect human melanocytes from UV-induced DNA damage and cytotoxicity,”FASEB J., 20:E888-E896 (2006); Bednarek, M. A., et al., “Cyclic analogsof α-melanocyte-stimulating hormone (αMSH) with high agonist potency andselectivity at human melanocortin receptor 1b,” Peptides, 29:1010-1017(2008); Koikov, L. N., et al., “Analogs of subnanomolar hMC1R agonistLK-184 [Ph(CH₂)₃CO-His-D-Phe-Arg-Trp-NH₂]. An additional binding sitewith the human melanocortin receptor 1?” Bioorg. Med. Chem. Lett.14:3997-4000 (2004); and Abdel-Malek, Z. A., et al., “Alpha-MSHtripeptide analogs activate the melanocortin 1 receptor and reduceUV-induced DNA damage in human melanocytes,” Pigment Cell Melanoma Res.22:635-44 (2009)).

Peptides or peptide-like compounds asserted to be specific for one ormore melanocortin receptors are disclosed in U.S. Pat. Nos. 5,576,290,5,674,839, 5,683,981, 5,714,576, 5,731,408, 6,051,555, 6,054,556,6,284,735, 6,350,430, 6,476,187, 6,534,503, 6,600,015, 6,613,874,6,693,165, 6,699,873, 6,887,846, 6,951,916, 7,008,925, 7,049,398,7,084,111, 7,176,279, 7,473,760, and 7,582,610; in U.S. published patentapplication Publication Nos. 2001/0056179, 2002/0143141, 2003/0064921,2003/0105024, 2003/0212002, 2004/0023859, 2005/0130901, 2005/0187164,2005/0239711, 2006/0105951, 2006/0111281, 2006/0293223, 2007/0027091,2007/0105759, 2007/0123453, 2007/0244054, 2008/0039387, and2009/0069242; and in international patent applications nos. WO 98/27113,WO 99/21571, WO 00/05263, WO 99/54358, WO 00/35952, WO 00/58361, WO01/30808, WO 01/52880, WO 01/74844, WO 01/85930, WO 01/90140, WO02/18437, WO 02/26774, WO 03/006604, WO 2004/046166, WO 2005/000338, WO2005/000339, WO 2005/000877, WO 2005/030797, WO 2005/060985,WO2006/048449, WO 2006/048450, WO 2006/048451, WO 2006/048452, WO2006/097526, WO 2007/008684, WO 2007/008704, WO 2007/009894 and WO2009/061411.

Notwithstanding the intense scientific and pharmaceutical interest inmelanocortin receptor-specific peptides, there remains a need for highlyselective and specific MCR-1 agonist peptides for use in pharmaceuticalapplications. It is against this background that the present inventionwas made.

BRIEF SUMMARY OF THE INVENTION

1. In one aspect, the invention provides a cyclic peptide of theformula:

including all enantiomers, stereoisomers or diastereoisomers thereof, ora pharmaceutically acceptable salt of any of the foregoing,

wherein:

-   -   R₄ is —(CH₂)_(w)—R₁₄;    -   R₇ is —(CH₂)_(w)—R₁₆;    -   R₉ is        -   —(CH₂)_(x)—C(═O)—NH—(CH₂)_(y)—,        -   —(CH₂)_(x)—NH—C(═O)—(CH₂)_(y)—,        -   —(CH₂)_(x)—C(═O)—(CH₂)_(z)—C(═O)—(CH₂)_(y)—,        -   —(CH₂)_(x)—C(═O)—NH—C(═O)—(CH₂)_(y)—,        -   —(CH₂)_(x)—NH—C(═O)—NH—(CH₂)_(y)—,        -   —(CH₂)_(x)—NH—C(═O)—(CH₂)_(z)—C(═O)—NH—(CH₂)_(y)—, or        -   —(CH₂)_(x)—S—S—(CH₂)_(y)—;    -   R₁₁ is H or a C₁ to C₁₇ acyl group, wherein the C₁ to C₁₇        comprises a linear or branched alkyl, cycloalkyl,        alkylcycloalkyl, aryl or alkylaryl;    -   R₁₄ is independently in each instance —N(R_(19a))(R_(19b)),        —NH—(CH₂)_(z)—N(R_(19a))(R_(19b)),        —NH—C(═NH)—N(R_(19a))(R_(19b)), —NH—C(═O)—N(R_(19a))(R_(19b)),        —O(R_(19a)), —(R_(19a))(R_(19b)), —S(═O)₂(R_(19a)),        —C(═O)—O(R_(19a)),

wherein any ring in R₁₄ is optionally substituted with one or more ringsubstituents, and when one or more substituents are present, are thesame or different and independently hydroxyl, halogen, sulfonamide,alkyl, —O-alkyl, aryl, —O-aryl, —C(═O)—OH, or—C(═O)—N(R_(19a))(R_(19b));

-   -   R₁₆ is —H, —N(R_(19a))(R_(19b)),        —NH—(CH₂)_(z)—N(R_(19a))(R_(19b)),        —NH—C(═NH)—N(R_(19a))(R_(19b)), —NH—C(═O)—N(R_(19a))(R_(19b)),        —O(R_(19a)), a linear or branched C₁ to C₁₇ alkyl chain,        —C(═O)—N(R_(19a))(R_(19b)), —S(═O)₂(R_(19a)),

wherein any ring is optionally substituted with one or more optionalring substituents, and when one or more substituents are present, arethe same or different and independently hydroxyl, halogen, sulfonamide,alkyl, —O-alkyl, aryl, aralkyl, O-aralkyl, or —O-aryl;

-   -   R₁₈ is —OH, —N(R_(19a))(R_(19b)), —N(R_(19a))(CH₂)_(w)        (C₁-C₇)cycloalkyl, or —O—(CH₂)_(w)—(C₁-C₇)cycloalkyl;    -   R_(19a) and R_(19b) are each independently H or a C₁ to C₄        linear or branched alkyl chain;    -   R₂₀ is naphthyl, optionally substituted with one or more        substituents independently selected from halo,        (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy,        (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro, nitrile, sulfonamide,        amino, monosubstituted amino, disubstituted amino, hydroxy,        carboxy, and alkoxy-carbonyl;    -   R_(21a), R_(21b) and R_(21c) are independently in each instance        hydrogen, halo, (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl,        (C₁-C₁₀)alkoxy, (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro,        nitrile, sulfonamide, amino, monosubstituted amino,        disubstituted amino, hydroxy, carboxy, or alkoxy-carbonyl;    -   R₂₂ is H or a C₁ to C₉ linear or branched alkyl, cycloalkyl,        alkylcycloalkyl, aryl or alkylaryl;    -   w is in each instance independently 0 to 5;    -   x is 1 to 5;    -   y is 1 to 5; and    -   z is in each instance independently 1 to 5.

The foregoing peptide comprises cyclic peptides wherein

-   -   R₄ is

-   -   R₉ is —(CH₂)_(x)—C(═O)—NH—(CH₂)_(y)— where x is 2 and y is 2, or        where x is 2 and y is 1    -   R₇ is

-   -   R₁₁ is a C₁ to C₁₇ acyl group, wherein the C₁ to C₁₇ comprises a        linear alkyl;    -   R₁₈ is —OH or —NH₂;    -   R_(21a), R_(21b) and R_(21c) are each H; and    -   R₂₂ is a C₁ to C₉ linear or branched alkyl.

Thus in one aspect the invention provides a cyclic peptide of theformula:

In another aspect the invention provides a cyclic peptide of theformula:

In another aspect the invention provides a cyclic peptide of theformula:

wherein

-   -   R₁₁ is a C₁ to C₁₇ acyl group, wherein the C₁ to C₁₇ comprises a        linear alkyl;    -   R₁₈ is —OH or —NH₂; and    -   R₂₂ is a C₁ to C₉ linear or branched alkyl.

In another aspect, the invention provides a cyclic peptide of theformula:

wherein

-   -   R₁₁ is a C₁ to C₁₇ acyl group, wherein the C₁ to C₁₇ comprises a        linear alkyl;    -   R₁₈ is —OH or —NH₂; and    -   R₂₂ is a C₁ to C₉ linear or branched alkyl.

In another aspect, the invention provides a cyclic peptide of theformula:

The invention further provides a cyclic peptide selected from the groupconsisting of:

-   -   Ac-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-D-Nal 1-NH₂ (SEQ ID NO:12),    -   Ac-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-Nal 2-NH₂ (SEQ ID NO:13),    -   Ac-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-D-Nal 2-NH₂ (SEQ ID NO:14),        and    -   Ac-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-Nal 1-NH₂ (SEQ ID NO:33),        or a pharmaceutically acceptable salt of any of the foregoing.

In another aspect, the present invention provides a melanocortinreceptor-specific peptide-based pharmaceutical composition for use intreatment of melanocortin receptor-mediated diseases, indications,conditions and syndromes.

In another aspect, the present invention provides a peptide-basedmelanocortin receptor-specific pharmaceutical, wherein the peptide is aselective MCR-1 ligand, for use in treatment of MCR-1 associateddisorders, diseases, indications, conditions and/or syndromes.

In another aspect, the present invention provides peptides which arespecific for melanocortin receptor MCR-1 and which are agonists.

In another aspect, the present invention provides a melanocortinreceptor-specific pharmaceutical for use in treatment whereinadministration of the treatment is via pulmonary administration.

In another aspect, the present invention provides specific MCR-1 cyclicpeptides that are effective over a significant dose range.

Yet another aspect of the present invention provides specific MCR-1cyclic peptides which, because of increased efficacy at low doses, maybe administered by delivery systems other than art conventionalintravenous, subcutaneous or intramuscular injection, including but notlimited to oral delivery systems, inhalation delivery systems, pulmonarydelivery systems, nasal delivery systems and mucous membrane deliverysystems.

Other aspects and novel features, and the further scope of applicabilityof the present invention will be set forth in part in the detaileddescription to follow, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The aspects of the invention may be realizedand attained by means of the instrumentalities and combinationsparticularly pointed out in the appended claims.

DETAILED DESCRIPTION OF THE INVENTION 1.0 Definitions

Before proceeding with the description of the invention, certain termsare defined as set forth herein.

In the sequences given for the peptides according to the presentinvention, the amino acid residues have their conventional meaning asgiven in Chapter 2400 of the Manual of Patent Examining Procedure,8^(th) Ed. Thus, “Nle” is norleucine, “Asp” is aspartic acid, “His” ishistidine, “Phe” is phenylalanine, “Arg” is arginine, “Trp” istryptophan, and “Lys” is lysine, and so on. It is to be understood that“D” isomers are designated by a “D-” before the three letter code oramino acid name, such that for example D-Phe is D-phenylalanine. Aminoacid residues not encompassed by the foregoing have the followingdefinitions:

Abbreviation Common Name Side Chain or Amino Acid Structure Aib alpha-aminoisobutyric acid

Cit citrulline

Dab diaminobutyric acid

Dap diaminopropionic acid

hGlu homoglutamic acid

Hyp hydroxyproline

Hyp(Bzl) O-benzyl- hydroxyproline

Nal 1 3- (1-naphthyl) alanine

Nal 2 3- (2-naphthyl) alanine

Nle norleucine

Orn ornithine

Pro(4-Bzl) 4-benzyl- proline

Pro(4-NH₂) 4-amino- proline

Sar sarcosine

An “α,α-disubstituted amino acid” means any α-amino acid having afurther substituent in the α-position, which substituent may be the sameas or different from the side chain moiety of the α-amino acid. Suitablesubstituents, in addition to the side chain moiety of the α-amino acid,include C₁ to C₆ linear or branched alkyl. Aib is an example of anα,α-disubstituted amino acid. While α,α-disubstituted amino acids can bereferred to using conventional L- and D-isomeric references, it is to beunderstood that such references are for convenience, and that where thesubstituents at the α-position are different, such amino acid caninterchangeably be referred to as an α,α-disubstituted amino acidderived from the L- or D-isomer, as appropriate, of a residue with thedesignated amino acid side chain moiety. Thus(S)-2-Amino-2-methyl-hexanoic acid can be referred to as either anα,α-disubstituted amino acid derived from L-Nle or as anα,α-disubstituted amino acid derived from D-Ala. Similarly, Aib can bereferred to as an α,α-disubstituted amino acid derived from Ala.Whenever an α,α-disubstituted amino acid is provided, it is to beunderstood as including all (R) and (S) configurations thereof. Whenevera claim or description herein refers to an “amino acid”, suchdesignation includes, but is not limited to, an “α,α-disubstituted aminoacid.”

An “N-substituted amino acid” means any amino acid wherein an amino acidside chain moiety is covalently bonded to the backbone amino group,including optionally where there are no substituents other than H in theα-carbon position. Sarcosine is an example of an N-substituted aminoacid. By way of example, sarcosine can be referred to as anN-substituted amino acid derivative of Ala, in that the amino acid sidechain moiety of sarcosine and Ala is the same, methyl. Whenever a claimor description herein refers to an “amino acid”, such designationincludes, but is not limited to, an “N-substituted amino acid.”

In certain instances groups may be substituted for an amino acid, suchas particularly use of a dicarboxylic acid in place of an amino acid.One particular dicarboxylic acid utilized herein is succinic acid,abbreviated as “Suc”, which has the structural formula

The term “alkane” includes linear or branched saturated hydrocarbons.Examples of linear alkane groups include methane, ethane, propane, andthe like. Examples of branched or substituted alkane groups includemethylbutane or dimethylbutane, methylpentane, dimethylpentane ortrimethylpentane, and the like. In general, any alkyl group may be asubstitutent of an alkane.

The term “alkene” includes unsaturated hydrocarbons that contain one ormore double carbon-carbon bonds. Examples of such alkene groups includeethylene, propene, and the like.

The term “alkenyl” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one double bond; examplesthereof include ethenyl, 2-propenyl, and the like.

The “alkyl” groups specified herein include those alkyl radicals of thedesignated length in either a straight or branched configuration.Examples of such alkyl radicals include methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl,isohexyl, and the like.

The term “alkyne” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one triple bond; examplesthereof include ethyne, propyne, butyne, and the like.

The term “aryl” includes a monocyclic or bicyclic aromatic hydrocarbonradical of 6 to 12 ring atoms, and optionally substituted independentlywith one or more substituents selected from alkyl, haloalkyl,cycloalkyl, alkoxy, alkythio, halo, nitro, acyl, cyano, amino,monosubstituted amino, disubstituted amino, hydroxy, carboxy, oralkoxy-carbonyl. Examples of an aryl group include phenyl, biphenyl,naphthyl, 1-naphthyl, and 2-naphthyl, derivatives thereof, and the like.

The term “aralkyl” includes a radical —R^(a)R^(b) where R^(a) is analkylene (a bivalent alkyl) group and R^(b) is an aryl group as definedabove. Examples of aralkyl groups include benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like.

The term “aliphatic” includes compounds with hydrocarbon chains, such asfor example alkanes, alkenes, alkynes, and derivatives thereof.

The term “acyl” includes a group R(C═O)—, where R is an organic group.An example is the acetyl group CH₃—C(═O)—, referred to herein as “Ac”.As used herein, R may comprise a C₁ to C₁₇ linear or branched alkyl,cycloalkyl, alkylcycloalkyl, aryl or alkylaryl.

A peptide or aliphatic moiety is “acylated” when an alkyl or substitutedalkyl group as defined above is bonded through one or more carbonyl{—(C═O)—} groups. A peptide is most usually acylated at the N-terminus.

An “omega amino aliphatic chain” includes an aliphatic moiety with aterminal amino group. Examples of omega amino aliphatic chains includeaminoheptanoyl and the amino acid side chain moieties of ornithine andlysine.

The term “heteroaryl” includes mono- and bicyclic aromatic ringscontaining from 1 to 4 heteroatoms selected from nitrogen, oxygen andsulfur. 5- or 6-membered heteroaryl are monocyclic heteroaromatic rings;examples thereof include thiazole, oxazole, thiophene, furan, pyrrole,imidazole, isoxazole, pyrazole, triazole, thiadiazole, tetrazole,oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and the like.Bicyclic heteroaromatic rings include, but are not limited to,benzothiadiazole, indole, benzothiophene, benzofuran, benzimidazole,benzisoxazole, benzothiazole, quinoline, benzotriazole, benzoxazole,isoquinoline, purine, furopyridine and thienopyridine.

An “amide” includes compounds that have a trivalent nitrogen attached toa carbonyl group (—C(═O)—NH₂), such as for example methylamide,ethylamide, propylamide, and the like.

An “imide” includes compounds containing an imido group(—C(═O)—NH—C(═O)—).

An “amine” includes compounds that contain an amino group (—NH₂).

A “nitrile” includes compounds that contain a (—CN) group bound to anorganic group.

The term “halogen” includes the halogen atoms fluorine, chlorine,bromine and iodine, and groups including one or more halogen atoms, suchas —CF₃ and the like.

The term “composition”, as in pharmaceutical composition, encompasses aproduct comprising the active ingredient(s), and the inert ingredient(s)that make up the carrier, as well as any product which results, directlyor indirectly, from combination, complexation or aggregation of any twoor more of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. Accordingly, the pharmaceutical compositionsutilized in the present invention encompass any composition made byadmixing an active ingredient and one or more pharmaceuticallyacceptable carriers.

By a melanocortin receptor “agonist” is meant an endogenous substance,drug substance or compound, including a compound such as the peptides ofthe present invention, which can interact with a melanocortin receptorand initiate a pharmacological response, including but not limited toadenyl cyclase activation, characteristic of the melanocortin receptor.For the present invention, a melanocortin receptor agonist which is anagonist at MCR-1 is preferred.

By “α-MSH” is meant the peptideAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (SEQ ID NO:2)and analogs and homologs thereof, including without limitationNDP-α-MSH.

By “NDP-α-MSH” is meant the peptideAc-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (SEQ IDNO:3) and analogs and homologs thereof.

By “EC₅₀” is meant the molar concentration of an agonist, including apartial agonist, which produced 50% of the maximum possible response forthat agonist. By way of example, a test compound which, at aconcentration of 72 nM, produces 50% of the maximum possible responsefor that compound as determined in a cAMP assay in an MCR-1 cellexpression system has an EC₅₀ of 72 nM. Unless otherwise specified, themolar concentration associated with an EC₅₀ determination is innanomoles per liter (nM).

By “Ki (nM)” is meant the equilibrium inhibitor dissociation constantrepresenting the molar concentration of a competing compound that bindsto half the binding sites of a receptor at equilibrium in the absence ofradioligand or other competitors. In general, the numeric value of theKi is inversely correlated to the affinity of the compound for thereceptor, such that if the Ki is low, the affinity is high. Ki may bedetermined using the equation of Cheng and Prusoff (Cheng Y., Prusoff W.H., Biochem. Pharmacol. 22: 3099-3108, 1973):

$K_{i} = \frac{{EC}_{50}}{1 + \frac{\lbrack{ligand}\rbrack}{K_{D}}}$

where “ligand” is the concentration of radioligand and K_(D) is aninverse measure of receptor affinity for the radioligand which produces50% receptor occupancy by the radioligand. Unless otherwise specified,the molar concentration associated with a Ki determination is in nM. Kimay be expressed in terms of specific receptors (e.g., MCR-1, MCR-3,MCR-4 or MCR-5), specific species (e.g., human or murine), and specificligands (e.g., α-MSH or NDP-α-MSH).

By “inhibition” is meant the percent attenuation, or decrease inreceptor binding, in a competitive inhibition assay compared to a knownstandard. Thus, by “inhibition at 1 μM (NDP-α-MSH)” is meant the percentdecrease in binding of NDP-α-MSH by addition of a determined amount ofthe compound to be tested, such as 1 μM of a test compound, such asunder the assay conditions hereafter described. By way of example, atest compound that does not inhibit binding of NDP-α-MSH has a 0%inhibition, and a test compound that completely inhibits binding ofNDP-α-MSH has a 100% inhibition. Typically, as described hereafter, aradio assay is used for competitive inhibition testing, such as withI¹²⁵-labeled NDP-α-MSH, or a lanthanide chelate fluorescent assay, suchas with Eu-NDP-α-MSH. However, other methods of testing competitiveinhibition are known, including use of label or tag systems other thanradioisotopes, and in general any method known in the art for testingcompetitive inhibition may be employed in this invention. It may thus beseen that “inhibition” is one measure to determine whether a testcompound attenuates binding of α-MSH to melanocortin receptors.

By “binding affinity” is meant the ability of a compound or drug to bindto its biological target, expressed herein as Ki (nM).

In general, “functional activity” is a measure of the signaling of areceptor, or measure of a change in receptor-associated signaling, suchas a melanocortin receptor, and in particular MCR-1 or hMCR-1, uponactivation by a compound. Melanocortin receptors initiate signaltransduction through activation of heterotrimeric G proteins. In oneaspect, melanocortin receptors signal through Gas, which catalyzesproduction of cAMP by adenylyl cyclase. Thus determination ofstimulation of adenylyl cyclase, such as determination of maximalstimulation of adenylyl cyclase, is one measure of functional activity,and is the primary measure exemplified herein. However, it is to beunderstood that alternative measures of functional activity may beemployed in the practice of this invention, and are specificallycontemplated and included within the scope of this invention. Thus, inone example intracellular free calcium may be measured, such as reportedby and using the methods disclosed in Mountjoy, K. G., et al.,“Melanocortin receptor-medicated mobilization of intracellular freecalcium in HEK293 cells,” Physiol. Genomics 5:11-19 (2001), or Kassack,M. U., et al., “Functional screening of G protein-coupled receptors bymeasuring intracellular calcium with a fluorescence microplate reader,”Biomol. Screening 7:233-246 (2002). It is also possible to measureactivation by measurement of the production of inositol triphosphate ordiacylglycerol from phosphatidylinositol 4,5-biphosphate, such as by useof radioassays. Yet another measure of functional activity is receptorinternalization, resulting from activation of regulatory pathways, suchas using the methods disclosed in Nickolls, S. A., et al., “Functionalselectivity of melanocortin 4 receptor peptide and nonpeptide agonists:evidence for ligand specific conformational states,” J. Pharm. Exper.Therapeutics 313:1281-1288 (2005). Yet another measure of functionalactivity is the exchange, and exchange rate, of nucleotides associatedwith activation of a G protein receptor, such as the exchange of GDP(guanosine diphosphate) for GTP (guanosine triphosphase) on the Gprotein α subunit, which may be measured by any number of means,including a radioassay using guanosine 5′-(γ-[³⁵S]thio)-triphosphate, asdisclosed in Manning, D. R., “Measures of efficacy using G proteins asendpoints: differential engagement of G proteins through singlereceptors,” Mol. Pharmacol. 62:451-452 (2002). Various gene-based assayshave been developed for measuring activation of G-coupled proteins, suchas those disclosed in Chen, W., et al., “A colorimetric assay frommeasuring activation of Gs- and Gq-coupled signaling pathways,” Anal.Biochem. 226:349-354 (1995); Kent, T. C., et al., “Development of ageneric dual-reporter gene assay for screening G-protein-coupledreceptors,” Biomol Screening 5:437-446 (2005); or Kotarsky, K., et al.,“Improved receptor gene assays used to identify ligands acting on orphanseven-transmembrane receptors,” Pharmacology & Toxicology 93:249-258(2003). The colorimetric assay of Chen et al. has been adapted for usein measuring melanocortin receptor activation, as disclosed in Hruby, V.J., et al., “Cyclic lactam α-melanocortin analogues ofAc-Nle⁴-cyclo[Asp⁵,D-Phe⁷,Lys¹⁰] α-melanocyte-stimulatinghormone-(4-10)-NH₂ with bulky aromatic amino acids at position 7 showshigh antagonist potency and selectivity at specific melanocortinreceptors,” J. Med. Chem., 38:3454-3461 (1995). In general, functionalactivity may be measured by any method, including methods of determiningactivation and/or signaling of a G-coupled receptor, and furtherincluding methods which may be hereafter developed or reported. Each ofthe foregoing articles, and the methods disclosed therein, isincorporated here by reference as if set forth in full.

The terms “treat,” “treating” and “treatment,” as used herein,contemplate an action that occurs while a patient is suffering from thespecified disease or disorder, which reduces the severity of the diseaseor disorder.

As used herein, the term “therapeutically effective amount” means theamount of a compound including a peptide of the invention that willelicit a biological or medical response in the mammal that is beingtreated by a medical doctor or other clinician.

As used herein, the term “prophylactically effective” or “preventive”means the amount of a compound including a peptide of the invention thatwill prevent or inhibit affliction or mitigate affliction of a mammalwith a medical condition that a medical doctor or other clinician istrying to prevent, inhibit, or mitigate before a patient begins tosuffer from the specified disease or disorder.

By “circulatory shock” is meant the general medical condition in whichorgans and/or tissues of the body of the subject, which subject may behuman or animal, are not receiving an adequate flow of blood.Circulatory shock includes conditions such as hypovolemic shock,cardiogenic shock, vasodilatory shock and the like. These conditions ordysfunctions in circulation can in turn have different causes, such asbacterial blood infection (septic shock or infectious), severe allergicreaction (anaphylactic shock), trauma (traumatic shock), severe bleedingor loss of blood (hemorrhagic shock), neurologic dysfunction causingabnormal opening of blood vessels (neurogenic shock) or endocrinerelated (endocrine shock). Circulatory shock can further result inischemia and ischemic damage to bodily organs, tissues, cells or parts.Upon reperfusion, or restoration of blood flow, ischemia-reperfusioninjury can occur, also resulting in damage to bodily organs, tissues, orcells.

By “inflammatory disease,” also sometimes called an “inflammatorycondition,” is meant a disease or condition characterized in part byinflammatory mechanisms, such as specific T lymphocyte reactions orantibody-antigen interactions causing the recruitment of inflammatorycells and endogenous mediator chemicals, including but not limited tocytokines, which mediator chemicals include but are not limited to oneor more of increased NF-κB activity, increased TNF-α production,increased IL-1 production and increased IL-6 production.

2.0 Clinical Indications and Utility

The compositions and methods disclosed herein can be used for bothmedical applications and animal husbandry or veterinary applications.Typically, the methods are used in humans, but may also be used in othermammals. The term “patient” denotes a mammalian individual, and is soused throughout the specification and in the claims. The primaryapplications of the present invention involve human patients, but thepresent invention may be applied to laboratory, farm, zoo, wildlife,pet, sport or other animals. Clinical indications and specific utilitiesinclude the following:

2.1 Inflammatory Diseases, Indications, Conditions and Syndromes

Peptides, compositions and methods of the present invention are directedtowards the treatment of inflammatory diseases and inflammatoryconditions in a subject. There are a number of inflammatory diseases andinflammatory conditions which may be so treated. In one aspect, theinflammatory condition results from a disease including a form ofarthritis, including but not limited to osteoarthritis, rheumatoidarthritis, septic arthritis, gout and pseudogout, juvenile idiopathicarthritis, Still's disease and ankylosing spondylitis, as well asarthritis secondary to other diseases, such as arthritis secondary tolupus erythematosus, Henoch-Schönlein purpura, psoriatic arthritis,reactive arthritis, haemochromatosis, hepatitis, Wegener'sgranulomatosis, vasculitis syndromes, Lyme disease, familialMediterranean fever, hyperimmunoglobulinemia D with recurrent fever, TNFreceptor-associated periodic syndrome and inflammatory bowel disease,including Crohn's disease and ulcerative colitis. In another aspect, theinflammatory condition results from a disease including a form ofinflammatory bowel disease, such as Crohn's disease, ulcerative colitis,collagenous colitis, lymphocytic colitis, ischemic colitis, diversioncolitis, Behçet's syndrome, infective colitis and indeterminate colitis.In another aspect, the inflammatory condition results from an autoimmunedisease, including but not limited to systemic syndromes such assystemic lupus erythematosus, Sjögren's syndrome, scleroderma,rheumatoid arthritis and polymyositis, or a syndrome affecting only alocal body system, such as the endocrine system (diabetes mellitus type1, Hashimoto's thyroiditis, Addison's disease, etc.), dermatologicsystem (pemphigus vulgaris), hematologic system (autoimmune hemolyticanemia), or neural system (multiple sclerosis). Thus autoimmune diseasesinclude, in addition to the general syndromes discussed above, suchdiseases and conditions as acute disseminated encephalomyelitis,Addison's disease, ankylosing spondylitis, antiphospholipid antibodysyndrome, aplastic anemia, autoimmune hepatitis, autoimmune oophoritis,celiac disease, Crohn's disease, gestational pemphigoid, Goodpasture'ssyndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease,idiopathic thrombocytopenic purpura, Kawasaki disease, lupuserythematosus, mixed connective tissue disease, multiple sclerosis,myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord'sthyroiditis, pemphigus, pernicious anaemia, primary biliary cirrhosis,Reiter's syndrome, Sjögren's syndrome, Takayasu's arteritis, temporalarteritis, autoimmune hemolytic anemia and Wegener's granulomatosis.

In another aspect, the inflammatory condition results from or is relatedto chronic obstructive pulmonary disease (COPD), also known as chronicobstructive airway diseases, including but not limited to diseasescharacterized by the pathological limitation of airflow in the airwaythat is not fully reversible, such as for example chronic bronchitis,emphysema, pneumoconiosis, pulmonary neoplasms and other lung disorders.Other inflammatory conditions include upper or lower airway diseases anddisorders, such as allergic asthma, non-allergic asthma, allergicrhinitis, vasomotor rhinitis, allergic conjunctivitis, non-allergicconjunctivitis, and the like, as well as airway diseases related toexternal toxins or substances, such as various forms of pneumoconiosis(coalworker's pneumoconiosis, asbestosis, silicosis, bauxite fibrosis,berylliosis, or siderosis), byssinosis or hypersensitivity pneumonitis(farmer's lung or bird fancier's lung). Other lung diseases involving aninflammatory condition include acute respiratory distress syndrome. Thepeptides and compositions of the present invention are of particularutility tor treatment of conditions wherein glucocorticoids are eitherineffectual or inadequate to bring about the desired pharmacologicalresponse, such as COPD, asthma in individuals who smoke, and otherconditions characterized, in whole or in part, by eosinophilaccumulation in the lung, neutrophil infiltration and activation,alveolar macrophage recruitment and activation, epithelial cellexpression of IL-8 or increased expression of TNF-α. For airway or lungdisorders, in one aspect the peptides of the present invention aredelivered systemically; in another aspect the peptides of the presentinvention are delivered locally, such as by inhalation administration.

In yet another aspect, the inflammatory condition results from or isrelated to some form of transplant-related condition or syndrome, suchas graft-versus-host disease, hyperacute rejection, acute rejection, orchronic rejection. Graft-versus-host disease is a common complication ofallogeneic bone marrow transplantation, but can occur with othertransplantations, and particularly those with T cells present in thegraft, either as contaminants or intentionally introduced. Hyperacute,acute or chronic rejection can occur with bodily organs such as kidneys,liver, pancreas, spleen, uterus, heart or lungs, as well astransplantation of bone, cornea, face, hand, penis or skin. In oneembodiment, a pharmaceutical composition including one or more of thepeptides of the present invention is given prophylactically to limit orprevent a transplant-related condition or syndrome, such as immediatelybefore, during or after transplantation of a bodily fluid, organ orpart. In another embodiment, the bodily fluid, organ or part beingtransplanted is perfused with a solution of a pharmaceutical compositionincluding one or more of the peptides of the present invention. In yetanother embodiment, one or more of the peptides of the present inventionare administered in conjunction with, combination with or series withone or more other agents for transplant rejection, such as calcineurininhibitors including cyclosporin or tacrolimus, mTOR inhibitorsincluding sirolimus or everolimus, anti-proliferatives includingazathioprine or mycophenolic acid, corticosteroids includingprednisolone or hydrocortisone, antibodies such as monoclonalanti-IL-2Rα receptor antibodies, basiliximab or daclizumab, orpolyclonal anti-T-cell antibodies such as anti-thymocyte globulin oranti-lymphocyte globulin.

2.2 Fibrotic and Sclerotic Diseases, Indications, Conditions andSyndromes

Peptides, compositions and methods of the present invention are directedtowards the treatment of fibrotic and sclerotic diseases, indications,conditions and syndromes in a subject. There are a number of fibroticand sclerotic diseases, indications, conditions and syndromes which maybe so treated. Fibrotic and sclerotic diseases, indications, conditionsand syndromes frequently include an inflammatory component, and thusmany may similarly be categorized as an inflammatory disease orcondition, and are listed in section 2.1 above. Fibrotic and scleroticdiseases and conditions, in addition to including an inflammatorycomponent, may also be idiopathic, toxic, hereditary and/orpharmacologically-induced disorders. In general, fibrotic disorders arecharacterized by excessive production of extracellular matrix, primarilytype I collagen, which may result in loss of organ function. It isbelieved, without wishing to be bound by theory, that agonism of MCR-1can result in suppression of transforming growth factor-β₁-inducedcollagen synthesis by human dermal fibroblasts, thereby providingtherapeutic and/or prophylactic benefit for fibrotic and scleroticdiseases, indications, conditions and syndromes. Representative fibroticand sclerotic diseases and conditions that can be so treated include,but are not limited to, localized scleroderma, systemic sclerosis,sclerodermic graft-versus-host disease of the skin, idiopathic lungfibrosis, bleomycin-induced lung fibrosis, cyclosporine-inducednephropathy, cirrhosis of the liver, hypertrophic scars, keloids and thelike.

2.3 Diseases Related to Increased Cytokine Expression and RelatedDiseases, Indications, Conditions and Syndromes

Expression of various cytokines is increased during an inflammatoryprocess, including an inflammatory process secondary to circulatoryshock, ischemia, reperfusion injury and the like. TNF-α is a pleiotropiccytokine produced mainly by macrophages, and also by other types ofcells. Other cytokines which increase during an inflammatory process,including an inflammatory process secondary to circulatory shock,ischemia, reperfusion injury and the like, include IL-1 and IL-6. Whilecytokines such as TNF-α have beneficial effects in many instances,significantly increased levels, such as secondary to circulatory shock,ischemia, reperfusion injury and the like, can have pathologicaleffects. In one aspect, reperfusion of hypoxic or ischemic tissues, suchas secondary to circulatory shock, results in inflammatory responses,including increased cytokine expression.

In one embodiment, the invention is directed to methods of using one ormore of the peptides of the present invention to decreasepro-inflammatory cytokine production and expression, includingdecreasing pro-inflammatory cytokine production and expression secondaryto circulatory shock, ischemia, reperfusion injury and the like. Thedecrease in pro-inflammatory cytokine production and expression,including without limitation one or more of TNF-α, IL-1 and IL-6, occursinstantaneously or within a short time period following administrationof a composition comprising one or more of the peptides of the presentinvention, preferably within at least about 40 minutes followingadministration, more preferably within 1-20 minutes, more preferablywithin 1-15 minutes, and most preferably within about 1-10 minutes.

In a related embodiment, the invention is directed to methods of usingone or more of the peptides of the present invention to increaseanti-inflammatory cytokine production and expression. The increase inanti-inflammatory cytokine production and expression, including withoutlimitation IL-10, occurs instantaneously or within a short time periodfollowing administration of a composition comprising one or more of thepeptides of the present invention, preferably within at least about 40minutes following administration, more preferably within 1-20 minutes,more preferably within 1-15 minutes, and most preferably within about1-10 minutes.

2.4 Dermatologic Diseases, Indications, Conditions and Syndromes

Peptides, compositions and methods of the present invention are furtherdirected towards the treatment of dermatologic and cosmetic diseases,indications, conditions and syndromes. In one aspect, peptides andcompositions of the present invention are MCR-1 agonists which stimulatemelanocytes and related cells to increase the level of melanin in theskin. By increasing the level of melanin in the skin, protection againstultraviolet radiation (UVR) and sunlight is afforded, includingprotection against phototoxicity and photosensitivity of the skin causedby UVR, sun and light.

In one aspect, peptides, compositions and methods of the presentinvention may be utilized for prophylactic and/or therapeutic treatmentof dermal diseases, indications, conditions and syndromes such as acnevulgaris (commonly referred to as acne), atopic dermatitis (commonlyreferred to as atopic eczema or eczema), polymorphous light eruption,psoriasis, rosacea, seborrheic dermatitis, vitiligo, porphyria,porphyria cutanea tarda, erythropoietic protoporphyria, solar urticaria,urticaria pigmentosa or xeroderma pigmentosum. In another aspect,peptides, compositions and methods of the present invention may beutilized to prevent, limit or treat photosensitive or photoresponsiveviral infections, such as herpes simplex virus (commonly referred to ascold sores and genital herpes depending on the site of infection), humanpapilloma virus and varicella zoster virus. In another aspect, peptides,compositions and methods of the present invention may be utilized toprevent, limit or treat cancers of the skin, including use inpre-cancerous conditions, and including use in actinic keratosis, basalcell carcinoma, melanoma or squamous cell carcinoma. In another aspect,peptides, compositions and methods of the present invention may beutilized to prevent or limit adverse effects of various therapies,including phototherapies, such as photodynamic therapy. In yet anotheraspect, peptides, compositions and methods of the present invention maybe utilized to induce a tan, to decrease hair graying or for similar andrelated purposes relating to increased melanin production.

Peptides of the present invention may be administered by any of avariety of means, including application directly to the skin such as bymeans of oils, ointments, creams, gels, salves and the like, or bysystemic administration, including by means of implants, such assubcutaneous dissolving implants.

2.5 Cytokine and/or Growth Factor Responsive Cancers

Certain cancers, such as mesothelioma, are reported to be very sensitiveto growth-promoting influences of cytokines and growth factors, and maybe treatable by means of peptides selective for MCR-1. Canania, A., etal., “Autocrine inhibitory influences of α-melanocyte-stimulatinghormone in malignant pleural mesothelioma,” J. Leukoc. Biol. 75:253-259(2004). Cancers that may be so treated include pleural mesothelioma,known to express mRHA for MCR-1 and the receptor protein, as well asother tumors that express MCR-1, including but not limited toadenocarcinoma, such as pulmonary adenocarcinoma.

2.6 Ocular Disease, Indications, Conditions and Syndromes

There are a number of ocular diseases, indications, conditions andsyndromes characterized by inflammation, including but not limited toincreased cytokine production. One example is dry eye disease, an oculardisease affecting approximately 10-20% of the population. This diseaseprogressively affects larger percentages of the population as it ages,with the majority of these patients being women. In addition, almosteveryone experiences ocular irritation, or the symptoms and/or signs ofdry eye as a condition, from time to time under certain circumstances,such as prolonged visual tasking (e.g., working on a computer), being ina dry environment, using medications that result in ocular drying and soon. In individuals suffering from dry eye, the protective layer of tearsthat normally protects the ocular surface is compromised, a result ofinsufficient or unhealthy production of one or more tear components.This can lead to exposure of the surface of the eye, ultimatelypromoting desiccation and damage of surface cells. Signs and symptoms ofdry eye include but are not limited to keratitis, conjunctival andcorneal staining, redness, blurry vision, decreased tear film break-uptime, decreased tear production, tear volume, and tear flow, increasedconjunctival redness, excess debris in the tear film, ocular dryness,ocular grittiness, ocular burning, foreign body sensation in the eye,excess tearing, photophobia, ocular stinging, refractive impairment,ocular sensitivity, and ocular irritation. Patients may experience oneor more of these symptoms. The excess tearing response may seemcounterintuitive, but it is a natural reflex response to the irritationand foreign body sensation caused by the dry eye. Some patients may alsoexperience ocular itching due to a combination of ocular allergy and dryeye symptoms.

There are many possible variables that can influence a patient's signsor symptoms of dry eye including levels of circulating hormones, variousautoimmune diseases (e.g., Sjogren's syndrome and systemic lupuserythematosus), ocular surgeries including PRK or LASIK, manymedications, environmental conditions, visual tasking such as computeruse, ocular fatigue, contact lens wear, and mechanical influences suchas corneal sensitivity, partial lid closure, surface irregularities(e.g., pterygium), and lid irregularities (e.g., ptosis,entropion/ectropion, pinguecula). Environments with low humidity, suchas those that cause dehydration, can exacerbate or cause dry eyesymptoms, such as sitting in a car with the defroster on or living in adry climate zone. In addition, visual tasking can exacerbate symptoms.Tasks that can greatly influence symptoms include watching TV or using acomputer for long periods of time where the blink rate is decreased.

Uveitis is an ocular disease involving inflammation of the middle layeror uvea of the eye, and may also be understood to include anyinflammatory process involving the interior of the eye. Uveitis includesanterior, intermediate, posterior and panuveitic forms, with themajority of uveitis cases anterior in location, involving inflammationof the iris and anterior chamber. This condition can occur as a singleepisode and subside with proper treatment or may take on a recurrent orchronic nature.

Symptoms include red eye, injected conjunctiva, pain and decreasedvision. Signs include dilated ciliary vessels, presence of cells andflare in the anterior chamber, and keratic precipitates on the posteriorsurface of the cornea. Intermediate uveitis includes inflammation andthe presence of inflammatory cells in the vitreous cavity, and posterioruveitis include the inflammation of the retina and choroid. Uveitis maybe secondary to any of a number of diseases and disorders, includingacute posterior multifocal placoid pigment epitheliopathy, ankylosingspondylitis, Behçet's disease, birdshot retinochoroidopathy,brucellosis, herpes simplex, herpes zoster, inflammatory bowel disease,juvenile rheumatoid arthritis, Kawasaki disease, leptospirosis, Lymedisease, multiple sclerosis, psoriatic arthritis, Reiter's syndrome,sarcoidosis, syphilis, systemic lupus erythematosus, toxocariasis,toxoplasmosis, tuberculosis, Vogt-Koyanagi-Harada syndrome, Whippledisease or polyarteritis nodosa.

In one embodiment, the invention is directed to methods of using one ormore of the peptides of the present invention for treatment of any ofthe foregoing ocular diseases, indications, conditions and syndromes.Such treatment may include treatment by means of eye drops, ointments,gels, washes, implants, plugs or other means and methods for deliveringone or more of the peptides of the present invention to an ocularsurface.

2.7 Ischemia and Related Diseases, Indications, Conditions and Syndromes

Ischemia refers to any decrease or stoppage in the blood supply to anybodily organ, tissue, cell, or part, particularly where that decrease orstoppage leads to or would likely lead to ischemic damage to the bodilyorgan, tissue, cell, or part. An “ischemic episode” refers to anytransient or permanent period of ischemia. Ischemia may result from anyconstriction or obstruction of the vasculature, or may result fromcirculatory shock, such as hemorrhagic shock, hypovolemic shock, or thelike. The decrease or lack of blood flow results in a decrease or lackof oxygen to the affected part of the body, and may also result in anincrease of inflammatory disease mediator chemicals such as variouscytokines and other substances. During certain surgical procedures suchas cardiac surgery and organ transplantation, the flow of blood isstopped temporarily and then resumed (reperfusion), resulting inischemia-reperfusion injury. During a heart attack, the blood thatsupplies the heart is stopped, also resulting in ischemia that canevolve into infarction. Current treatment to relieve heart attacksrequires reperfusion of the ischemic area of the heart, such as by usingthrombolytic drugs or coronary angioplasty.

The invention has particular application in prevention of injury due torenal ischemia, including lung injury secondary to renal ischemia,preventing or limiting ischemic heart injuries subsequent to amyocardial infarction, preventing or limiting ischemic brain injuriessubsequent to a cardiovascular injury, including without limitationmyocardial infarction, stroke or the like. Neuroprotection is providedby administration of a composition of the invention to a patient withcerebral ischemia or stroke, particularly patients who are concurrentlyhypotensive. The invention has further particular application inpreventing or limiting ischemic organ damage in organ transplant,including transplant of the heart, kidney, liver, lungs, pancreas orsmall intestine. In one aspect, the pharmaceutical composition of thepresent invention may be utilized for perfusion of a transplant organ,which perfusion may be prior to, during or subsequent to transplant ofthe organ.

In one embodiment, the invention is directed to methods of using one ormore of the peptides of the present invention to protect the heart,brain or other organs of a patient against injury caused by ischemia.The protective effect against ischemia occurs instantaneously or withina short time period following administration of a composition comprisingone or more of the peptides of the present invention, preferably withinat least about 40 minutes following administration, more preferablywithin 1-20 minutes, more preferably within 1-15 minutes, and mostpreferably within about 1-10 minutes.

Ischemia may also results from any of a variety of diseases orconditions, and in one embodiment the invention is directed to methodsof using one or more of the peptides of the present invention to protectthe organs of a patient against injury resulting from ischemia, whichischemia is caused by a disease or condition. Such disease or conditionmay include, by way of example and not limitation, atheroscleroticdiseases such as atheromata with thrombosis, embolism from the heart orfrom blood vessel from any organ, vasospasm, hypotension due to heartdisease, hypotension due to systemic disease including infection orallergic reactions, or hypotension resulting from administration,ingestion or other exposure to one or more toxic compounds or drugs.Ischemia may also be secondary ischemia, and in another embodiment theinvention is directed to methods of using one or more of the peptides ofthe present invention to protect the organs of a patient against injuryresulting from secondary ischemia. Such secondary ischemia may besecondary to a disease or condition such diabetes mellitus,hyperlipidemia, hyperlipoproteinemia, dyslipidemia Buerger's disease,also called thromboangiitis obliterans, Takayasu's arteritis, arteritistemporalis, Kawasaki disease, also called lymph node syndrome,mucocutaneous node disease, infantile polyarteritis, cardiovascularsyphilis, and various connective tissue diseases and disorders.

2.8 Ischemia-Reperfusion Injury and Related Diseases, Indications,Conditions and Syndromes

Ischemia-reperfusion is the interruption of blood flow to bodily tissueand the subsequent and often abrupt restoration of blood flow to thetissue. While restoration of blood flow following ischemia is essentialto preserve functional tissue, the reperfusion itself is known to beharmful to the tissue. Both ischemia and reperfusion are known to beimportant contributors to tissue necrosis. Several mechanisms appear toplay a causative role in the generation of tissue damage associated withischemia-reperfusion injury.

Various methods of limiting reperfusion injury have been described, suchas induced hypothermia, controlled reperfusion, and ischemicpreconditioning. Induced hypothermia is the induction of moderatehypothermia, thought to suppress many of the chemical reactionsassociated with reperfusion injury. Controlled reperfusion refers tocontrolling the initial period of reperfusion by reperfusing the tissueat a low pressure using blood that has been modified to be hyperosmolar,alkalotic, and substrate-enriched. Ischemic preconditioning is thepurposeful causing of short ischemic events to have protective effect byslowing cell metabolism during a longer ischemic event. Although thesetreatments may be useful in surgical settings (e.g., before or afterplanned heart surgery), they are not possible in emergency settings.

The invention has particular application in preventing or limiting theseverity of renal reperfusion injury, including lung injury secondary torenal reperfusion, preventing or limiting reperfusion heart injuriessubsequent to a myocardial infarction, preventing or limitingreperfusion brain injuries subsequent to a cardiovascular injury,including without limitation myocardial infarction, stroke or the like.The invention has further particular application in preventing orlimiting reperfusion organ damage in organ transplant, includingtransplant of the heart, kidney, liver, lungs, pancreas or smallintestine. In one aspect, the pharmaceutical composition of the presentinvention may be utilized for perfusion of a transplant organ, whichperfusion may be prior to, during or subsequent to transplant of theorgan.

In one embodiment, the invention is directed to methods of using one ormore of the peptides of the present invention to protect the heart,brain or other organs of a patient against injury caused byischemia-reperfusion injury, including injury caused by or duringreperfusion. The protective effect against ischemia-reperfusion injuryoccurs instantaneously or within a short time period followingadministration of a composition comprising one or more of the peptidesof the present invention, preferably within at least about 40 minutesfollowing administration, more preferably within 1-20 minutes, morepreferably within 1-15 minutes, and most preferably within about 1-10minutes.

2.9 Circulatory Shock and Related Diseases, Indications, Conditions andSyndromes

Peptides, compositions and methods of the present invention may beemployed for the treatment of circulatory shock in a subject. Thecompositions and methods provided herein may be employed to treat StageI shock, Stage II shock or Stage III shock. In one particularembodiment, the methods of the present invention are used to treat theinitial stage of shock, which initial stage of shock is characterized bycardiac output insufficient to meet the body's metabolic needs, but nototherwise low enough to produce significant symptoms. The patient may beanxious and alert, with increased respiration.

By “Stage I shock,” also sometimes called “compensated shock” or“non-progressive shock,” is meant a condition which occurs when the bodydetects decreased blood flow or perfusion and begins to activate one ormore of several reactive mechanisms to restore perfusion or direct bloodflow to the most vital body organs. Stage I shock can be asymptomatic,but may also include, but is not limited to, symptoms such as low bloodflow or perfusion, rapid or increased heart rate, shallow or irregularbreathing, hypotension, hypertension, pallor and cyanosis.

By “Stage II shock,” also sometimes called “decompensated shock” or“progressive shock,” is mean a condition which occurs when thecompensatory mechanisms of the body begin to fail and organ perfusioncannot be restored to normal or maintained. Symptoms of Stage II shockinclude, but are not limited to, confusion, anxiety, disorientation andother mental disturbances indicating a lack of oxygen to the brain,chest pains, increased heart rate, oliguria, multiple organ dysfunction,falling blood pressure (hypotension), rapid breathing, weakness andpupil dilation.

By “Stage III shock,” also sometimes called “irreversible shock,” ismeant a condition which occurs after the state of decreased perfusion orblood flow has existed to such an extent that the organs and tissues ofthe body are permanently affected. Such symptoms include, but are notlimited to, multiple organ failure, kidney failure, coma, blood poolingin the extremities and death.

The invention provides compositions for use and methods of treating orpreventing hemorrhagic shock in a patient, which include administering acomposition including one or more of the peptides of the presentinvention to a patient diagnosed as suffering from blood loss. The bloodloss may, but need not, be measured as a percentage of the subject'sblood volume, such as, for example, a blood loss of greater than about15% total blood volume, or greater than 20%, 25%, 30%, 35%, 40%, or 50%of the subject's total volume. Alternatively, the blood loss may, butneed not, be measured in terms of a drop in blood volume in any amountsufficient to cause hemorrhagic shock in a particular subject, such as,for example, a loss of about 750 mL, 1000 mL, of about 1500 mL, or ofabout 2000 mL or more in a human subject. The blood loss may also bemeasured in terms of a drop in systolic blood pressure, such as, forexample, a drop in systolic blood pressure that is about 20 mm Hg, 30 mmHg, 40 mm Hg, 50 mm Hg, 60 mm Hg, 70 mm Hg, 80 mm Hg, 90 mm Hg or 100 mmHg or more than 100 mm Hg lower than the subject's normal systolic bloodpressure. In particular embodiments, the subject is undergoing or hasundergone a medical procedure, such as, but not limited to, surgery, atransfusion or child birth. In other particular embodiments, the subjecthas suffered a traumatic injury, such as, but not limited to, resultingfrom a motor vehicle accident, from an industrial injury, or from agunshot wound.

In additional embodiments of the present invention, the compositions andmethods are used to treat cardiogenic shock, hypovolemic shock andvasodilatory shock, each of which can be in any of the aforementionedstages of shock. In one particular embodiment of the present invention,the methods are used to treat cardiogenic shock. Cardiogenic shock is,generally speaking, low blood flow or perfusion that is caused by heartmalfunction where the heart does not pump adequate blood. Causes caninclude any condition that interferes with ventricular filling oremptying, such as, but not limited to, embolism, ischemia, regurgitationand valve malfunction. In another particular embodiment of the presentinvention, the methods are used to treat vasodilatory shock.Vasodilatory shock is caused by severe venous or arteriolar dilation,which results in inadequate blood flow. Several known causes contributeto vasodilatory shock including, but not limited to, cerebral trauma,drug or poison toxicity, anaphylaxis, liver failure, bacteremia andsepsis. In another more particular embodiment of the present invention,the methods are used to treat shock resulting from sepsis or bacteremia.In an even more particular embodiment, the compositions and methods areused to treat septic shock or bacteremic shock in Stage I, II or Ill. Inyet another embodiment, the compositions and methods of the presentinvention are used to treat hypovolemic shock. Hypovolemic shock is,generally speaking, decreased intravascular volume, which decrease inintravascular volume can be relative or absolute. Hemorrhage fromconditions such as, but not limited to, ulcers, gastrointestinal injury,trauma, accidents, surgery, and aneurysm may cause hypovolemic shock;but loss of other body fluids may also cause hypovolemic shock. Forinstance, renal fluid loss, intravascular fluid loss, water or otherperitoneal fluid loss may contribute to hypovolemic shock. In oneparticular embodiment of the present invention, the compositions andmethods, including administration of one or more of the peptides of thepresent invention, are used to treat hypovolemic shock. In an even moreparticular embodiment, the compositions and methods are used to treathypovolemic shock in Stage I, Stage II or Stage III.

Circulatory shock, including hemorrhagic shock, may also result frompartially controlled or uncontrolled bleeding within one or moreinternal organs or vessels of a patient. Bleeding may result from anycause, including by way of example from a ruptured aneurysm, dissectedaorta, an ulcer, trauma or other gastrointestinal bleeding. In someinstances the patient exhibits signs of circulatory shock orhypovolemia, which may include hypotension, but the source of internalbleeding is unknown.

In one embodiment, the invention is directed to methods of using one ormore of the peptides of the present invention to protect the heart,brain or other organs of a patient against injury caused by circulatoryshock. The protective effect against circulatory shock occursinstantaneously or within a short time period following administrationof a composition comprising one or more of the peptides of the presentinvention, preferably within at least about 40 minutes followingadministration, more preferably within 1-20 minutes, more preferablywithin 1-15 minutes, and most preferably within about 1-10 minutes.

2.10 Targeted Imaging and Cytotoxic Therapy for Melanoma and OtherIndications

Peptides, compositions and methods of the present invention may beemployed for imaging melanoma and other cancers or diseases orconditions characterized, in part, by relatively high expression ofMCR-1, such as by diagnostic imaging using a radionuclide in combinationwith a peptide of the present invention. For diagnostic imaging,typically a peptide of the present invention is conjugated toradionuclide by use of a linker, such as a cross-linking agent thatcouples the peptide of the present invention to a radionuclide. Theradionuclide is preferably a gamma emitter that may be imaged using agamma detector or camera, such as single photon emission computedtomography, or is a positron emitter that may be imaged using positronemission tomography. Gamma emitters that may be so employed include^(99m)Tc, ¹¹¹In, ¹²³I and ⁶⁷Ga, among others. Positron emitters that maybe so employed include ¹¹C, ¹³N, ¹⁵O and ¹⁸F.

In a related aspect, peptides, compositions and methods of the presentinvention may be employed for cytotoxic therapy of melanoma, othercancers or diseases or conditions characterized, in part, by relativelyhigh expression of MCR-1, such as by utilizing a chemotherapeutic agent,including toxins, or radiation therapeutic agent, in combination with apeptide of the present invention. Chemotherapeutic agents include anyantineoplastic drug or chemical, such as for example alkylating agents,antimetabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, and other antitumor agents. Non-limiting examples ofalkylating agents include cisplatin, carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide; examplesof antimetabolites include azathioprine and mercaptopurine; examples ofanthracyclines include daunorubicin, doxorubicin, epirubicin,idarubicin, valrubicin and mitoxantrone; examples of plant alkaloidsinclude vinca alkaloids such as vincristine, vinblastine, vinorelbineand vindesine and taxanes such as paclitaxel and docetaxel; examples oftopoisomerase inhibitors include camptothecins such as irinotecan andtopotecan and type II topoisomerases such as amsacrine, etoposide,etoposide phosphate and teniposide. However, any agent suitable for usein targeted cytotoxic therapy may be so employed. Non-limiting examplesof radiation therapeutic agents that may be so employed include ¹³¹I,¹²⁵I, ²¹¹At, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ¹⁵³Sm, ²¹²Bi and ³²P, among others.

Diagnostic imaging or cytotoxic therapy agents may be incorporated intoa peptide of the present invention, for example such as by use of ¹¹C,¹³N, ¹⁵O, among others, in place of nonradioactive isotopes; may belinked directly to a peptide of the present invention, such as forexample by halogenation or other direct complexation methods; or may belinked indirectly to a peptide of the present invention, such asconjugation by means of a linker or chelation unit. Linker units arewell known in the art, and include, but are not limited to,chemically-linked conjugates including at least one disulfide bond,thioether bond or covalent bond between free reactive groups.Representative cross-linking and conjugating reagents are disclosed inU.S. Pat. Nos. 7,169,603, 7,820,164 and 5,443,816 and US Publication No.2009/0297444, among others, incorporated herein by reference.

3.0 Combination Therapy for Certain Indications

The peptides, compositions and methods of the present invention may beused for treatment of any of the foregoing diseases, indications,conditions or syndromes, or any disease, indication, condition orsyndrome which is MCR-1 mediated or responsive, by administration incombination with one or more other pharmaceutically active compounds.Such combination administration may be by means of a single dosage formwhich includes both a peptide of the present invention and one moreother pharmaceutically active compounds, such single dosage formincluding a tablet, capsule, spray, inhalation powder, injectable liquidor the like. Alternatively, combination administration may be by meansof administration of two different dosage forms, with one dosage formcontaining a peptide of the present invention, and the other dosage formincluding another pharmaceutically active compound. In this instance,the dosage forms may be the same or different. The term “coadminister”indicates that each of at least two compounds in the combination therapyare administered during a time frame wherein the respective periods ofbiological activity or effects overlap. Thus the term includessequential as well as concurrent administration of compounds where onecompound is one or more of the peptides of the present invention. Ifmore than one compound is coadministered, the routes of administrationof the two or more compounds need not be the same. Without meaning tolimit combination therapies, the following exemplifies certaincombination therapies which may be employed.

3.1 Combination Therapy with Anti-Inflammatory Agents

For the treatment of inflammation-related diseases, indications,conditions and syndromes, peptides of the present invention may be usedin combination therapy, including by means of coadministration, with oneor more anti-inflammatory agents. One class of anti-inflammatory agentis glucocorticoids, including but not limited to cortisone, includingcortisone acetate, hydrocortisone, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, prednisone, fludrocortisone acetate, deoxycorticosteroneacetate and aldosterone. Other anti-inflammatory agents that may be usedin combination therapy, including by means of coadministration, includeaspirin, non-steroidal antiinflammatory drugs (NSAIDs) (such asibuprofen and naproxin), TNF-α inhibitors (such as tenidap and rapamycinor derivatives thereof), or TNF-α antagonists (e.g., infliximab,OR1384), cyclooxygenase inhibitors (i.e., COX-1 and/or COX-2 inhibitorssuch as Naproxen® or Celebrex®), CTLA4-lg agonists/antagonists, CD40ligand antagonists, IMPDH inhibitors, such as mycophenolate (CellCept®),integrin antagonists, alpha-4 beta-7 integrin antagonists, cell adhesioninhibitors, interferon gamma antagonists, ICAM-1, prostaglandinsynthesis inhibitors, budesonide, clofazimine, p38 mitogen-activatedprotein kinase inhibitors, protein tyrosine kinase (PTK) inhibitors, IKKinhibitors, therapies for the treatment of irritable bowel syndrome(e.g., Zelmac® and Maxi-K® openers such as those disclosed in U.S. Pat.No. 6,184,231), or other NF-κB inhibitors, such as corticosteroids,calphostin, CSAIDs, 4-substituted imidazo [1,2-A]quinoxalines asdisclosed in U.S. Pat. No. 4,200,750; Interleukin-10, salicylates,nitric oxide, and other immunosuppressants; and nuclear translocationinhibitors, such as deoxyspergualin (DSG).

3.2 Combination Therapy with Phosphodiesterase Inhibitors

For certain applications and indications, it is desirable to increaseproduction of and maintain levels of cyclic adenoise 3′,5′ monophosphate(cAMP), a nucleotide messenger associated with inflammatory cellactivity. Peptides of the present invention increase intracellularlevels of cAMP, and can be coadministered with compounds or substancesthat inhibit the degradation of cAMP. cAMP is hydrolyzed to an inactiveform by phosphodiesterase (PDE); compounds or substances that inhibitPDE may thereby result in maintenance of and/or an increase in availablecAMP. A class of compounds known as PDE inhibitors has been extensivelystudied for use in treatment of inflammatory diseases, such as asthma,COPD and acute respiratory distress syndrome. Preferred are inhibitorsof PDE type 1, 2, 3, 4, 7, 8, 10 or 11; in one aspect this includescAMP-PDE inhibitors that are selective PDE type 4 inhibitors orinhibitors having selectivity for one particular type of PDE 4isoenzyme, such as, by way of example, rolipram, cilomilast, ibudilast,and piclamilast. In general, the methods and compositions of thisinvention may comprise use of one or more cAMP-PDE inhibitors describedin one or more of the following U.S. patents or patent applications,each of which is incorporated herein by reference: U.S. Pat. Appl. No.20090221664, “Pharmaceutical Compositions of Muscarinic ReceptorAntagonists”; U.S. Pat. Appl. No. 20090054382, “Compositions ofPhosphodiesterase Type IV Inhibitors”; U.S. Pat. Appl. No. 20090017036,“Pharmaceutical Compositions for Treatment of Respiratory andGastrointestinal Disorders”; U.S. Pat. Appl. No. 20080292562,“Medicaments for Inhalation Comprising PDE IV Inhibitors andEnantiomerically Pure Glycopyrrolate Salts”; U.S. Pat. Appl. No.20080085858, “Pharmaceutical Composition”; U.S. Pat. Appl. No.20080045718, “Process and intermediates for the synthesis of2-(quinolin-5-yl)-4,5 disubstituted-azole derivatives”; U.S. Pat. Appl.No. 20070287689, “Therapeutic and/or Preventive Agents for Chronic SkinDiseases”; U.S. Pat. Appl. No. 20060239927, “Drug for airwayadministration”; U.S. Pat. No. 7,544,675, “Chemical compounds with dualactivity, processes for their preparation and pharmaceuticalcompositions”; U.S. Pat. No. 7,459,451, “Pyrazolopyridine derivatives”;U.S. Pat. No. 7,317,009, “Pyrrolopyridazine derivatives”; U.S. Pat. No.7,312,328, “Benzoylpyridazines”; U.S. Pat. No. 7,153,854,“Pyrrolopyridazine derivatives”; U.S. Pat. No. 7,115,623, “PDE IVinhibitors”; U.S. Pat. No. 6,924,292, “Furoisoquinoline derivatives,process for producing the same and use thereof”; U.S. Pat. No.6,872,382, “Use of selective PDE IV inhibitors to treat dry eyedisorders”; U.S. Pat. No. 6,765,095, “2,3-disubstituted pyridinederivative, process for the preparation thereof, pharmaceuticalcomposition containing the same, and intermediate therefor”; U.S. Pat.No. 6,740,662, “Naphthyridine derivatives”; U.S. Pat. No. 6,683,186,“2,3-Disubstituted pyridine derivative, process for the preparationthereof, pharmaceutical composition containing the same, andintermediate therefor”; U.S. Pat. No. 6,656,959, “PDE IV inhibitingpyridine derivatives”; U.S. Pat. No. 6,642,250,“1,8-naphthyridin-2(1H)-one derivatives”; U.S. Pat. No. 6,555,557,“2,3-disubstituted pyridine derivatives, process for the preparationthereof, drug compositions containing the same and intermediates for thepreparation”; U.S. Pat. No. 6,440,979, “Aryl isoguanines”; U.S. Pat. No.6,436,965, “PDE IV inhibiting amides, compositions and methods oftreatment”; U.S. Pat. No. 6,417,190, “Tricyclic nitrogen heterocycles asPDE IV inhibitors”; U.S. Pat. No. 6,413,975, “Purine derivatives havingphosphodiesterase IV inhibition activity”; U.S. Pat. No. 6,403,805,“1,3-dihydro-1-(phenylalkyl)-2H-imidazol-2-one derivatives”; U.S. Pat.No. 6,372,770, “Benzoxazoles”; U.S. Pat. No. 6,365,606, “6,5-fusedaromatic ring systems having enhanced phosphodiesterase IV inhibitoryactivity”; U.S. Pat. No. 6,310,205, “Hypoxathine compounds”; U.S. Pat.No. 6,306,583, “Human brain phosphodiesterase”; U.S. Pat. No. 6,268,373,“Trisubstituted thioxanthines”; U.S. Pat. No. 6,248,769,“Phenyl-triazole compounds for PDE-IV inhibition”; U.S. Pat. No.6,248,768, “Benzimidazole derivatives and pharmacologically acceptablesalts thereof”; U.S. Pat. No. 6,248,746, “3-(arylalkyl) xanthines”; U.S.Pat. Nos. 6,211,222 and 6,127,398, “Substituted indazole derivatives andrelated compounds”; U.S. Pat. No. 6,211,203,“Benzofuran-4-carboxamides”; U.S. Pat. No. 6,200,993, “Heterosubstitutedpyridine derivatives as PDE4 inhibitors”; U.S. Pat. No. 6,191,138,“Phenanthridines”; U.S. Pat. No. 6,180,650, “Heterosubstituted pyridinederivatives as PDE4 inhibitors”; U.S. Pat. No. 6,136,821, “Naphthyridinederivatives”; U.S. Pat. No. 6,054,475, “Substituteddihydrobenzofuran-based phosphodiesterase 4 Inhibitors useful fortreating airway disorders”; U.S. Pat. No. 6,043,263,“(2,3-dihydrobenzofuranyl)-thiazoles as phosphodiesterase inhibitors”;U.S. Pat. No. 6,011,037, “Thiazole derivatives withphosphodiesterase-inhibiting action”; U.S. Pat. No. 5,972,927,“Diazepinoindoles as phosphodiesterase 4 inhibitors”; U.S. Pat. No.5,919,801, “N-substituted piperidines as PDE4 inhibitors”; U.S. Pat. No.6,204,275, “PDE IV Inhibiting compounds, compositions and methods oftreatment”; U.S. Pat. No. 6,143,782, “Anti-inflammatory and anti-asthmatreatment with reduced side effects”; U.S. Pat. No. 6,103,749, “Arylimidazole compounds having phosphodiesterase IV activity”; U.S. Pat. No.6,096,768, “Compounds containing phenyl linked to aryl or heteroaryl byan aliphatic or heteroatom containing linking group”; U.S. Pat. No.6,075,016, “6,5-fused aromatic ring systems having enhancedphosphodiesterase IV inhibitory activity”; U.S. Pat. No. 6,040,447,“Purine compounds having PDE IV inhibitory activity and methods ofsynthesis”; U.S. Pat. No. 6,034,089, “Aryl thiophene derivatives as PDEIV inhibitors”; U.S. Pat. No. 6,020,339, “Aryl furan derivatives as PDEIV inhibitors”; U.S. Pat. No. 5,935,978, “Compounds containing phenyllinked to aryl or heteroaryl by an aliphatic or heteroatom containinglinking group”; U.S. Pat. No. 5,935,977, “Substituted vinyl pyridinederivative and drugs containing same”; U.S. Pat. No. 5,840,724,“Compounds containing phenyl linked to aryl or heteroaryl by analiphatic or heteroatom containing linking group”; U.S. Pat. No.5,710,170, “Tri-aryl ethane derivatives as PDE IV inhibitors”; U.S. Pat.No. 5,710,160, “Diphenyl pyridyl ethane derivatives as PDE IVinhibitors”; U.S. Pat. No. 5,698,711, “Compounds containing phenyllinked to aryl or heteroaryl by an aliphatic or heteroatom containinglinking group”; U.S. Pat. No. 5,691,376, “Substituted biphenylderivatives”; U.S. Pat. No. 5,679,696, “Compounds containing phenyllinked to aryl or heteroaryl by an aliphatic or heteroatom containinglinking group”; U.S. Pat. No. 5,665,737, “Substituted benzoxazoles”;U.S. Pat. No. 5,650,444, “Substituted biphenyl derivatives”; U.S. Pat.No. 5,616,614, “Naphthylalkylamines”; U.S. Pat. No. 5,541,219,“1-Alkoxy-2-(alkoxy or cycloalkoxy)-4-(cyclothio-alkyl orcyclothioalkenyl)benzenes as inhibitors of cyclic AMP phosphodiesteraseand tumor necrosis factor”; U.S. Pat. No. 5,502,072, “Substitutedoxindoles”; U.S. Pat. No. 5,466,697,“8-phenyl-1,6-naphthyri-dine-5-ones”; U.S. Pat. No. 5,459,151, “N-acylsubstituted phenyl piperidines as bronchodilators and anti-inflammatoryagents”; U.S. Pat. No. 5,393,788, “Phenylalkyl oxamides”; U.S. Pat. No.5,356,923, “1-hydroxy-4(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidoneand anti-hypertensive use thereof”; U.S. Pat. No. 5,250,700, “Phenylpyrazolidinones as bronchodilators and anti-inflammatory agents”; U.S.Pat. No. 5,191,084, “Phenyl pyrazolidinones as bronchodilators andanti-inflammatory agents”; U.S. Pat. No. 5,124,455, “Oxime carbamatesand oxime carbonates as bronchodilators and anti-inflammatory agents”;U.S. Pat. No. 6,180,791, “Synthesis of 8-substituted xanthines”; U.S.Pat. No. 6,057,369, “Substituted (aryl, heteroaryl, arylmethyl orheteroarylmethyl)hydroxamic acid compounds”; U.S. Pat. No. 5,541,219,“1-Alkoxy-2-(alkoxy or cycloalkoxy)-4-(cyclothioalkyl orcyclothioalkenyl)benzenes as inhibitors of cyclic AMP phosphodiesteraseand tumor necrosis factor”; U.S. Pat. No. 5,362,915, “Phenyl substitutedcycloalkenyl compounds useful as PDE IV inhibitors”; U.S. Pat. No.6,040,329, “Substituted indazole analogs”; U.S. Pat. No. 5,958,953,“Substituted indazole derivatives”; U.S. Pat. No. 6,090,817,“Phenylpyridine derivatives useful as phosphodiesterase inhibitors”;U.S. Pat. No. 5,922,740, “Heterocyclylcarbonyl substitutedbenzofuranylureas”; U.S. Pat. No. 5,866,571, “9-substituted2-2-n-alkoxyphenyl)-purin-6-ones-”; U.S. Pat. No. 5,861,404,“2,9-disubstituted purin-6-ones”; U.S. Pat. No. 5,861,396, “Purin-6-onederivatives”; U.S. Pat. No. 5,721,238, “2,8-disubstitutedquinazolinones”; U.S. Pat. No. 5,723,463, “Pyrido 3,2-Pyrazinones withAnti-asthmatic action and Processes for their Manufacture”; and U.S.Pat. No. 5,596,013, “Dihydro pyrazolopyrroles.”

3.3 Combination Therapy in Ocular Indications

For ocular indications, an ophthalmic dosage form may include one ormore active ingredients in addition to one or more of the peptides ofthe present invention, such as for example artificial tear components,topical corticosteroids, non-steroidal anti-inflammatory drugs, orcalcineurin inhibitors such as cyclosporine-A (Restasis®—Allergan). Itis also possible that coadministration includes administration of one ormore additional compounds given separately from a peptide of the presentinvention, such as separate administration of an ophthalmic dosage formincluding an artificial tear component, a topical corticosteroid, anon-steroidal anti-inflammatory drugs, a calcineurin inhibitor such acyclosporine-A, or a combination of any of the foregoing.

Combination ophthalmic solutions may be employed, including specificallysolutions including more than one active pharmaceutical ingredient. Inone aspect, a non-steroidal anti-inflammatory drug (NSAID) is employedin combination with a peptide of the present invention. NSAIDs suitablefor use in combination ophthalmic solutions include agents, their estersand pharmaceutically acceptable salts thereof that inhibit thecycloxygenase (COX)-1 and/or -2 enzyme, including but not limited topropionic acid compounds such as naproxen, flurbiprofen, oxaprozin,ibuprofen, ketoprofen, fenoprofen; ketorolac tromethamine; acetic acidderivatives such as sulindac, indomethacin, and etodolac; phenylaceticacids such as diclofenac, bromfenac, and suprofen; arylacetic prodrugssuch as nepafenac, and amfenac; salicyclic acids, such as aspirin,salsalate, diflunisal, choline magnesium trisalicylate; para-aminophenolderivatives such as acetaminophen; naphthylalkanones such as nabumetone;enolic acid derivatives such as piroxicam and meloxicam; femanates suchas mefenamic acid, meclofenamate and flufenamic acid; pyrroleaceticacids such as tolmetin; and pyrazolones such as phenylbutazone; andCOX-2 selective inhibitors such as celecoxib, valdecoxib, parecoxib,etoricoxib, and luaricoxib. The ophthalmic solutions may additionallycomprise other active ingredients, including, but not limited to,vasoconstrictors, anti-allergenic agents, anti-infectives, steroids,anesthetics, anti-inflammatories, analgesics, dry eye treatment agents(e.g. secretagogues, mucomimetics, polymers, lipids, antioxidants), andthe like, or be administered in conjunction (simultaneously orsequentially) with pharmaceutical compositions comprising other activeingredients, including, but not limited to, vasoconstrictors,anti-allergenic agents, anti-infectives, steroids, anesthetics,anti-inflammatories, analgesics, dry eye treatment agents (e.g.secretagogues, mucomimetics, polymers, lipids, antioxidants), and thelike.

3.4 Combination Therapy in Shock-Related Indications

The methods of treating or preventing circulatory shock of the presentinvention also relate to coadministering one or more substances to thesubject in addition to one or more of the peptides of the presentinvention. For example, one or more of the peptides of the presentinvention may be coadministered with androstenetriol, androstenediol orderivatives thereof, various vasopressin agonists, or otherpharmaceutically active substances, such as catecholamines or other aadrenergic agonists, α₂ adrenergic agonists, β adrenergic agonists or β₂adrenergic agonists, including but not limited to epinephrine,norepinephrine, dopamine, isoproterenol, vasopressin and dobutamine.Alternatively, one or more of the peptides of the present invention maybe coadministered with fluids or other substances that are capable ofalleviating, attenuating, preventing or removing symptoms in a subjectsuffering from, exhibiting the symptoms of, or at risk of suffering fromhypovolemic shock, vasodilatory shock or cardiogenic shock. Types offluid that can be coadministered with one or more of the peptides of thepresent invention should be specific to the circumstances surroundingthe particular subject that is suffering from, exhibiting the symptomsof, or at risk of suffering from shock. For example, fluids that may becoadministered with one or more of the peptides of the present inventioninclude, but are not limited to, salt solutions—such as sodium chlorideand sodium bicarbonate—as well as whole blood, synthetic bloodsubstitutes, plasma, serum, serum albumin and colloid solutions. Colloidsolutions include, but are not limited to, solutions containinghetastarch, albumin or plasma. In one particular embodiment of thepresent invention, fluids such as one or more of salt solutions,colloidal solutions, whole blood, synthetic blood substitutes, plasma orserum are coadministered with one or more of the peptides of the presentinvention in patients suffering from or exhibiting the symptoms of ahypovolemic shock, such as hemorrhagic shock.

Particular embodiments of the coadministration methods of the presentinvention include methods of performing a transfusion in a subject, withthe transfusion methods comprising providing blood or synthetic bloodsubstitutes that comprise one or more of the peptides of the presentinvention to a subject. The blood used in the transfusion methods can bewhole blood, synthetic blood substitutes, or any fractionated portion ofwhole blood, such as plasma, serum, or red blood cells.

4.0 Methods of Administration and Use

The method of administration and use varies depending upon thecharacteristic of specific peptides of the present invention, thedisease, indication, condition or syndrome to be treated, and otherfactors known to those in the art. In general, any method ofadministration and use known in the art or hereafter developed may beemployed with the peptides of the present invention. Without limitingthe foregoing, the following methods of administration and use havespecific application.

4.1 Inhalation Use

In one aspect, a composition including one or more peptides of thepresent invention is formulated for administration to the respiratorytract, such as in the form of an aerosol or solution for a nebulizer, oras a microfine powder for insufflation or inhalation (e.g., topically tothe lung and/or airways), alone or in combination with one or more inertcarriers or additional active pharmaceutical ingredients, and in theform of a solution, a suspension, an aerosol or a dry powderformulation. See generally, Cryan, S.-A., “Carrier-based strategies fortargeting protein and peptide drugs to the lungs,” The AAPS Journal7:E20-41 (2005). In general, the peptides of the present invention maybe used the devices, formulations, compositions and means described inone or more of the following U.S. patents or patent applications, eachof which is incorporated herein by reference: U.S. Pat. Appl. No.20090241949, “Dry powder inhalation system”; U.S. Pat. Appl. No.20080066741, “Methods and systems of delivering medication viainhalation”; U.S. Pat. Appl. No. 20070298116, “Amorphous, spray-driedpowders having a reduced moisture content and a high long termstability”; U.S. Pat. Appl. No. 20070140976, “Aqueous inhalationpharmaceutical composition”; U.S. Pat. Appl. No. 20060054166,“Inhalation nebulizer”; U.S. Pat. Appl. No. 20050211244, “Dry powderpreparations”; U.S. Pat. Appl. No. 20050123509. “Modulating chargedensity to produce improvements in the characteristics of spray-driedproteins”; U.S. Pat. Appl. No. 20040241232, “Dry powder medicamentformulations”; U.S. Pat. No. 7,582,284, “Particulate materials”; U.S.Pat. No. 7,481,212, “Increased dosage metered dose inhaler”; U.S. Pat.No. 7,387,794, “Preparation of powder agglomerate”; U.S. Pat. No.7,258,873, “Preservation of bioactive materials by spray drying”; U.S.Pat. No. 7,186,401, “Dry powder for inhalation”; U.S. Pat. No.7,143,764, “Inhalation device”; U.S. Pat. No. 7,022,311, “Powderyinhalational preparations and process for producing the same”; U.S. Pat.No. 6,962,151, “Inhalation nebulizer”; U.S. Pat. No. 6,907,880,“Inhalation device”; U.S. Pat. No. 6,881,398, “Therapeutic dry powderpreparation”; U.S. Pat. No. 6,698,425, “Powder inhaler”; U.S. Pat. No.6,655,380, “Inhalation device”; U.S. Pat. No. 6,645,466, “Dry powder forinhalation”; U.S. Pat. No. 6,632,456, “Compositions for inhalation”;U.S. Pat. No. 6,610,272, “Medicinal aerosol formulation”; U.S. Pat. No.6,596,261, “Method of administering a medicinal aerosol formulation”;U.S. Pat. No. 6,585,957, “Medicinal aerosol formulation”; U.S. Pat. No.6,582,729, “Powered pharmaceutical formulations having improveddispersibility”; U.S. Pat. No. 6,572,893, “Systems and processes forspray drying hydrophobic drugs with hydrophilic excipients”; U.S. Pat.No. 6,551,578, “Modulated release particles for aerosol delivery”; U.S.Pat. No. 6,520,179, “Inhalation device”; U.S. Pat. No. 6,518,239, “Drypowder compositions having improved dispersivity”; U.S. Pat. No.6,503,481, “Compositions for aerosolization and inhalation”; U.S. Pat.No. 6,358,530, “Powdered pharmaceutical formulations having improveddispersibility”; U.S. Pat. No. 6,325,061, “Inhalation device”; U.S. Pat.No. 6,257,232, “Inhalation device”; U.S. Pat. No. 6,187,344, “Powderedpharmaceutical formulations having improved dispersibility”; U.S. Pat.No. 6,116,237, “Methods of dry powder inhalation”; U.S. Pat. No.5,934,272, “Device and method of creating aerosolized mist ofrespiratory drug”; and, U.S. Pat. No. 5,558,085, “Intrapulmonarydelivery of peptide drugs”.

Thus the composition may be a dry powder composition for topicaldelivery to the lung by inhalation. Typically the composition wouldcontain a powder mix for inhalation of a peptide of the presentinvention and a suitable powder base, diluent or carrier substance suchas lactose, glucose, dextran, mannitol or another sugar or starch. Thecomposition may be used in any of a variety of dry powder devices, suchas a reservoir dry powder inhaler, a multi-dose dry powder inhaler, or ametered dose inhaler. The composition may include additional excipients,such as an alcohol, a surfactant, a lubricant, an anti-oxidant or astabilizing agent. Suitable propellants include hydrocarbon,chlorofluorocarbon and hydrofluoroalkane propellants, or mixtures of anysuch propellants.

Inhalation solutions also can be formulated in a liquefied propellantfor aerosol delivery, such as with a pressurized metered dose inhaler.In yet another formulation, solutions may be in the form of a nebulisedaqueous suspension or solution, with or without a suitable pH ortonicity adjustment, either as a single dose or multidose device.

4.2 Subcutaneous Injection Use

In one aspect, a composition including one or more peptides of thepresent invention is formulated for subcutaneous injection, and asubcutaneous injection is given one or more times each day, preferablyprior to a meal, more preferably between about one and about three hoursprior to a mean. In another aspect, the composition is formulated as aninjectable sustained release formulation. In one embodiment, a peptideof the present invention is formulated with a polyethylene glycol, suchas polyethylene glycol 3350, and optionally one or more additionalexcipients and preservatives, including but not limited to excipientssuch as salts, polysorbate 80, sodium hydroxide or hydrochloric acid toadjust pH, and the like. In another embodiment a peptide of the presentinvention is formulated with a poly(ortho ester), which may be anauto-catalyzed poly(ortho ester) with any of a variable percentage oflactic acid in the polymeric backbone, and optionally one or moreadditional excipients. In one embodiment poly (D,L-lactide-co-glycolide)polymer (PLGA polymer) is employed, preferably a PLGA polymer with ahydrophilic end group, such as PLGA RG502H from Boehringer Ingelheim,Inc. (Ingelheim, Germany). Such formulations may be made, for example,by combining a peptide of the present invention in a suitable solvent,such as methanol, with a solution of PLGA in methylene chloride, andadding thereto a continuous phase solution of polyvinyl alcohol undersuitable mixing conditions in a reactor. In general, any of a number ofinjectable and biodegradable polymers, which are preferably alsoadhesive polymers, may be employed in a sustained release injectableformulation. The teachings of U.S. Pat. Nos. 4,938,763, 6,432,438, and6,673,767, and the biodegradable polymers and methods of formulationdisclosed therein, are incorporated here by reference. The formulationmay be such that an injection is required on a weekly, monthly or otherperiodic basis, depending on the concentration and amount of peptide,the biodegradation rate of the polymer, and other factors known to thoseof skill in the art.

4.3 Methods of Administration and Use for Circulatory Shock and RelatedDiseases, Indications, Conditions and Syndromes

In yet another aspect, the invention includes methods which optionallyinclude monitoring the subject for symptoms of circulatory shock bothbefore and after administration of a pharmaceutical compositionincluding one or more of the peptides of the present invention. Thus asubject may be administered one or more of the peptides of the presentinvention by one of the methods of the invention after suffering aninjury likely to induce circulatory shock but prior to the manifestationof overt symptoms of cardiovascular shock, including prior tomanifestation of circulatory shock in Stage I, Stage II or Stage III.

When administration is for the purpose of treatment, one or more of thepeptides of the present invention are provided at, or after the onsetof, a symptom of shock. The therapeutic administration of one or more ofthe peptides of the present invention may further serve to attenuate anysymptom, or prevent additional symptoms from arising. Whenadministration is for the purposes of preventing shock (“prophylacticadministration”), one or more of the peptides of the present inventionare provided in advance of any visible or detectable symptom. Theprophylactic administration of one or more of the peptides of thepresent invention serve to attenuate subsequently arising symptoms orprevent symptoms from arising altogether. The route of administration ofone or more of the peptides of the present invention include, but arenot limited to, topical, transdermal, intranasal, pulmonary, vaginal,rectal, oral, subcutaneous, intravenous, intraarterial, intramuscular,intraosseous, intraperitoneal, epidural and intrathecal.

4.4 Methods of Administration and Use for Prophylactic Therapy

The invention also relates to methods of preventing undesired cytokineexpression by administering a therapeutically effective amount of one ormore of the peptides of the present invention to the subject, prior toor immediately at the onset of the first symptoms. As used herein, theterm “prevent,” as it relates to shock, indicates that a substance ofthe present invention is administered to a subject to prohibit one ormore symptoms of shock from detectably appearing or to attenuate theeffects of one or more symptoms of shock. The term “prevent” alsoencompasses prohibiting or limiting excessive or undesired cytokineexpression, such as with a “cytokine storm”. Thus a subject may be“pretreated,” such as a subject in a surgical setting, by using thesubstances of the present invention to prevent undesired cytokineexpression or shock from arising. The phrase “preventing theprogression,” as it relates to shock, is used to mean a proceduredesigned to prohibit the detectable appearance of one or more additionalsymptoms of shock in a patient already exhibiting one or more symptomsof shock, and is also used to mean prohibiting the already-presentsymptoms of shock from worsening in the subject. The symptoms of shockthat are included in preventative methods of the present inventioninclude, but are not limited to, such symptoms of shock as highlightedherein, such as tachycardia, shallow or erratic breathing and death. Asubject that is at risk of shock may be recognized based upon thespecific circumstances surrounding a subject. Similarly, a patient witha bacterial or viral infection and exhibiting a fever or low bloodpressure may also be at risk of excessive cytokine expression, shock oran inflammatory disease or condition.

In additional embodiments of the present invention, the methods are usedto prevent cardiogenic shock, hypovolemic shock and vasodilatory shock,each of which can be in any of the three aforementioned stages of shock.In one particular embodiment of the present invention, the methods areused to prevent cardiogenic shock. In another particular embodiment ofthe present invention, the methods are used to prevent vasodilatoryshock. In another more particular embodiment of the present invention,the methods are used to prevent shock resulting from sepsis orbacteremia. In an even more particular embodiment, the methods are usedto prevent septic shock or bacteremic shock in Stage I, II or III shock.In yet another embodiment, the methods of the present invention are usedto prevent hypovolemic shock.

Similar to the methods of treating shock described herein, oneembodiment of the methods of preventing shock of the present inventioncomprises coadministering another substance with one or more of thepeptides of the present invention or a derivative thereof. The scope ofthe invention is not limited by the identity of the substance which maybe coadministered with one or more of the peptides of the presentinvention to prevent shock. For example, one or more of the peptides ofthe present invention may be coadministered with androstenetriol,androstenediol or derivatives thereof, various vasopressin agonists, orother pharmaceutically active substances, such as catecholamines orother a adrenergic agonists, α₂ adrenergic agonists, β adrenergicagonists or β₂ adrenergic agonists, including but not limited toepinephrine, norepinephrine, dopamine, isoproterenol, vasopressin anddobutamine, to prevent shock.

Alternatively, one or more of the peptides of the present invention maybe coadministered with fluids or other substances that are capable ofpreventing or removing symptoms in a subject at risk of suffering fromhypovolemic shock, vasodilatory shock or cardiogenic shock. The types offluid that can be coadministered with one or more of the peptides of thepresent invention to prevent shock should be specific to thecircumstances surrounding the particular subject that is at risk ofsuffering from shock. For example, fluids that may be coadministeredwith one or more of the peptides of the present invention include, butare not limited to, salt solutions—such as sodium chloride and sodiumbicarbonate—as well as whole blood, synthetic blood substitutes, plasma,serum, serum albumin and colloid solutions. Colloid solutions include,but are not limited to, solutions containing hetastarch, albumin orplasma. In one particular embodiment of the present invention, fluidsincluding one or more of salt solutions, colloidal solutions, wholeblood, synthetic blood substitutes, plasma or serum are coadministeredwith one or more of the peptides of the present invention or aderivative thereof in subjects at risk of suffering a hypovolemic shock,such as hemorrhagic shock.

4.5 Methods of Administration and Use for Inflammation RelatedApplications, Diseases, Indications, Conditions and Syndromes

In yet another aspect, the invention includes methods which optionallyinclude monitoring the subject for signs or symptoms of inflammation,inflammatory diseases or inflammatory conditions both before and afteradministration of one or more of the peptides of the present invention.Thus a subject may be administered one or more of the peptides of thepresent invention by one of the methods of the invention after beingdiagnosed with a condition, disease or syndrome likely to induce aninflammatory response, but prior to the manifestation of overt symptomsof inflammation, inflammatory disease or inflammatory condition. Methodsof treating or preventing inflammation, inflammatory diseases orinflammatory conditions described herein comprise administering atherapeutically effective amount of one or more of the peptides of thepresent invention to a subject. As used herein, the term “administer”and “administering” are used to mean introducing at least one compoundinto a subject. When administration is for the purpose of treatment, thesubstance is provided at, or after the onset of, a sign or symptom ofinflammation, inflammatory disease or inflammatory condition. Thetherapeutic administration of this substance serves to attenuate anysymptom, or prevent additional symptoms from arising. Whenadministration is prophylactic administration for the purposes ofpreventing or limiting inflammation, inflammatory disease or aninflammatory condition, a pharmaceutical composition including one ormore of the peptides of the present invention is provided in advance ofany visible or detectable symptom. The prophylactic administration ofone or more of the peptides of the present invention serves to attenuatesubsequently arising symptoms or prevent symptoms from arisingaltogether. The route of administration of one or more of the peptidesof the present invention include, but are not limited to, topical,transdermal, intranasal, pulmonary, vaginal, rectal, oral, subcutaneous,intravenous, intraarterial, intramuscular, intraosseous,intraperitoneal, epidural and intrathecal.

4.6 Methods of Administration and Use for Ocular Diseases, Indications,Conditions and Syndromes

For ocular applications, in one aspect one or more of the peptides ofthe present invention are formulated in an ophthalmic dosage form andadministered in the form of eye drops, eye washes or by means of otherocular delivery systems. Emulsions, ointments, gels, ocular inserts,biodegradable ocular inserts, liposomes, microparticles, nanoparticles,nanospheres or ion pairing formulations may also be employed, which may,in some instances, result in increasing the ocular residence times of apeptide of the present invention. In one embodiment, the ophthalmicformulation is a solution that includes between about 0.0000001% andabout 5% (w/v) of a peptide of the present invention or a salt thereof,alternatively between about 0.000001% and about 0.2% (w/v) of a peptideof the present invention or a salt thereof, or alternatively betweenabout 0.00001% and about 0.2% (w/v) of a peptide of the presentinvention or a salt thereof.

5.0 Methods of Making

In general, the peptides of the present invention may be synthesized bysolid-phase synthesis and purified according to methods known in theart. Any of a number of well-known procedures utilizing a variety ofresins and reagents may be used to prepare the peptides of the presentinvention.

The cyclic peptides of the present invention may be readily synthesizedby known conventional procedures for the formation of a peptide linkagebetween amino acids. Such conventional procedures include, for example,any solution phase procedure permitting a condensation between the freealpha amino group of an amino acid or residue thereof having itscarboxyl group and other reactive groups protected and the free primarycarboxyl group of another amino acid or residue thereof having its aminogroup or other reactive groups protected. In a preferred conventionalprocedure, the cyclic peptides of the present invention may besynthesized by solid-phase synthesis and purified according to methodsknown in the art. Any of a number of well-known procedures utilizing avariety of resins and reagents may be used to prepare the peptides ofthe present invention.

The process for synthesizing the cyclic peptides may be carried out by aprocedure whereby each amino acid in the desired sequence is added oneat a time in succession to another amino acid or residue thereof or by aprocedure whereby peptide fragments with the desired amino acid sequenceare first synthesized conventionally and then condensed to provide thedesired peptide. The resulting peptide is then cyclized to yield acyclic peptide of the invention.

Solid phase peptide synthesis methods are well known and practiced inthe art. In such methods the synthesis of peptides of the invention canbe carried out by sequentially incorporating the desired amino acidresidues one at a time into the growing peptide chain according to thegeneral principles of solid phase methods. These methods are disclosedin numerous references, including Merrifield, R. B., “Solid phasesynthesis (Nobel lecture),” Angew Chem 24:799-810 (1985) and Barany etal., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E.and Meienhofer, J., Eds. Academic Press 1-284 (1980).

In chemical syntheses of peptides, reactive side chain groups of thevarious amino acid residues are protected with suitable protectinggroups, which prevent a chemical reaction from occurring at that siteuntil the protecting group is removed. Also common is the protection ofthe alpha amino group of an amino acid residue or fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the alpha amino protecting group to allow a subsequent reaction totake place at that site. Specific protecting groups have been disclosedand are known in solid phase synthesis methods and solution phasesynthesis methods.

Alpha amino groups may be protected by a suitable protecting group,including a urethane-type protecting group, such as benzyloxycarbonyl(Z) and substituted benzyloxycarbonyl, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz)and aliphatic urethane-type protecting groups, such ast-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,isopropoxycarbonyl, and allyloxycarbonyl (Alloc). Fmoc is preferred foralpha amino protection.

Guanidino groups may be protected by a suitable protecting group, suchas nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc),adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) andBoc. Pbf and Pmc are preferred protecting groups for Arg.

The peptides of the invention described herein were prepared using solidphase synthesis, such as by means of a Symphony Multiplex PeptideSynthesizer (Rainin Instrument Company) automated peptide synthesizer,using programming modules as provided by the manufacturer and followingthe protocols set forth in the manufacturer's manual.

Solid phase synthesis is commenced from the C-terminal end of thepeptide by coupling a protected alpha amino acid to a suitable resin.Such starting material is prepared by attaching an alpha amino-protectedamino acid by an amide linkage to 9-Fmoc-aminoxanthen-3-yloxy-Merrifieldresin (Sieber Amide resin) or to4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink Amideresin), by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin,a 2-chlorotrityl chloride resin or an oxime resin, or by other meanswell known in the art. The resins are carried through repetitive cyclesas necessary to add amino acids sequentially. The alpha amino Fmocprotecting groups are removed under basic conditions. Piperidine,piperazine, diethylamine, or morpholine (20-40% v/v) inN,N-dimethylformamide (DMF) may be used for this purpose.

Following removal of the alpha amino protecting group, the subsequentprotected amino acids are coupled stepwise in the desired order toobtain an intermediate, protected peptide-resin. The activating reagentsused for coupling of the amino acids in the solid phase synthesis of thepeptides are well known in the art. After the peptide is synthesized, ifdesired, the orthogonally protected side chain protecting groups may beremoved using methods well known in the art for further derivatizationof the peptide.

Typically, orthogonal protecting groups are used as appropriate. Forexample, the peptides of the invention contain multiple amino acids withan amino group-containing side chain. In one aspect, an Allyl-Allocprotection scheme is employed with the amino acids forming a lactambridge through their side chains, and orthogonal protecting groups,cleavable under different reactive conditions, use for other amino acidswith amino group-containing side chains. Thus, for example,Fmoc-Lys(Alloc)-OH, Fmoc-Orn(Alloc)-OH, Fmoc-Dap(Alloc)-OH,Fmoc-Dab(Alloc)-OH, Fmoc-Asp(OAII)-OH or Fmoc-Glu(OAII)-OH amino acidscan be employed for the positions forming a lactam bridge uponcyclization, while other amino acids with amino group-containing sidechains have a different and orthogonal protecting group, such as withFmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Dab(Boc)-OH or the like. Otherprotecting groups may be similarly employed; by way of example and notlimitation, Mtt/OPp (4-methyltrityl/2-phenylisopropyl) can be employedwith the side chains forming a lactam bridge upon cyclization, withorthogonal protecting groups being utilized for other positions that arenot cleavable using conditions suitable for cleavage of Mtt/OPp.

Reactive groups in a peptide can be selectively modified, either duringsolid phase synthesis or after removal from the resin. For example,peptides can be modified to obtain N-terminus modifications, such asacetylation, while on resin, or may be removed from the resin by use ofa cleaving reagent and then modified. Similarly, methods for modifyingside chains of amino acids are well known to those skilled in the art ofpeptide synthesis. The choice of modifications made to reactive groupspresent on the peptide will be determined, in part, by thecharacteristics that are desired in the peptide.

In the peptides of the present invention, in one embodiment theN-terminus group is modified by introduction of an N-acetyl group. Inone aspect, a method is employed wherein after removal of the protectinggroup at the N-terminal, the resin-bound peptide is reacted with aceticanhydride in N,N-dimethylformamide (DMF) in the presence of an organicbase, such as pyridine. Other methods of N-terminus acetylation areknown in the art, including solution phase acetylation, and may beemployed.

The peptide can, in one embodiment, be cyclized prior to cleavage fromthe peptide resin. For cyclization through reactive side chain moieties,the desired side chains are deprotected, and the peptide suspended in asuitable solvent and a cyclic coupling agent added. Suitable solventsinclude, for example DMF, dichloromethane (DCM) or1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling reagents include,for example, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU),benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP),benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate(PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TATU), 2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU) orN,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCCI/HOBt).Coupling is conventionally initiated by use of a suitable base, such asN,N-diisopropylethylamine (DIPEA), sym-collidine or N-methylmorpholine(NMM).

For peptides with a non-lactam cyclic bridge, such as peptidescontaining the bridge:

—(CH₂)_(x)—NH—C(═O)—(CH₂)_(z)—C(═O)—NH—(CH₂)_(y)—,

where x, y and z are each independently 1 to 5, the peptides may be madeusing solid phase synthesis employing a side-chain protected diamineamino acid for the positions to be cyclized. Particularly preferred insuch positions are Dap, Dab or Lys, preferably with an amine protectinggroup such as Alloc, Mtt, Mmt (methoxytrityl), Dde(1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene))ethyl), ivDde(1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl) or anyother orthogonally cleavable protecting group. Typically, one side chainprotecting group is removed first, such as removal of Mtt using 2% TFAin dichloromethane. Following washing of the resin, the resultingresin-bound unprotected amine is acylated, such as with a 0.5 M solutionof a cyclic anhydride such as succinic anhydride or glutaric anhydridein dichloromethane/pyridine 1:1. Following additional wash steps, theorthogonally cleavable protecting group of the second diamino amino acidis cleaved, such as removal of Alloc usingtetrakis(triphenylphosphine)palladium(0) and phenyl silane indichloromethane. After washing with dichloromethane and DMF theresin-bound peptide is cyclized using standard coupling reagents such asTBTU and a base. Alternatively, an ivDde protected resin-bound diaminoamino acid can be deprotected using a solution of 5% of hydrazine inDMF, and after washing with DMF the resulting resin bound amine caneither be acylated with a cyclic anhydride or can be cyclized with aresin bound carboxylic acid.

The cyclized peptides can then be cleaved from solid phase, using anysuitable reagent, such as ethylamine in DCM or various combinations ofagents, such as trifluoroacetic acid (TFA), tri-isopropylsilane (TIS),dimethoxybenezene (DMB), water and the like. The resulting crude peptideis dried and remaining amino acid side chain protecting groups, if any,are cleaved using any suitable reagent, such as (TFA) in the presence ofwater, TIS, 2-mercaptopethane (ME), and/or 1,2-ethanedithiol (EDT). Thefinal product is precipitated by adding cold ether and collected byfiltration. Final purification is by reverse phase high performanceliquid chromatography (RP-HPLC), using a suitable column, such as a C₁₈column, or other methods of separation or purification, such as methodsbased on the size or charge of the peptide, can also be employed. Oncepurified, the peptide can be characterized by any number of methods,such as high performance liquid chromatograph (HPLC), amino acidanalysis, mass spectrometry, and the like.

For peptides of the present invention which have a C-terminussubstituted amide derivative or N-alkyl group, synthesis may proceed bysolid phase synthesis commenced from the C-terminal end of the peptideby coupling a protected alpha amino acid to a suitable resin. Suchmethods for preparing substituted amide derivatives on solid-phase havebeen described in the art. See, for example, Barn, D. R., et al.,“Synthesis of an array of amides by aluminum chloride assisted cleavageon resin bound esters,” Tetrahedron Letters, 37:3213-3216 (1996);DeGrado, W. F. and Kaiser E. T., “Solid-phase synthesis of protectedpeptides on a polymer bound oxime: Preparation of segments comprisingthe sequences of a cytotoxic 26-peptide analogue,” J. Org. Chem.,47:3258-3261 (1982). Such a starting material can be prepared byattaching an alpha amino-protected amino acid by an ester linkage to ap-benzyloxybenzyl alcohol (Wang) resin or an oxime resin by well knownmeans. The peptide chain is grown with the desired sequence of aminoacids, the peptide cyclized and the peptide-resin treated with asolution of appropriate amine (such as methyl amine, dimethyl amine,ethylamine, and so on). Peptides employing a p-benzyloxybenzyl alcohol(Wang) resin may be cleaved from resin by aluminum chloride in DCM, andpeptides employing an oxime resin may be cleaved by DCM. Another methodto prepare a peptide with a C-terminus substituted amide is to attach analkyl amine by reductive amination to a formyl resin, such as4-(4-Formyl-3-methoxyphenoxy)butyryl-AM resin (FMPB AM resin), and thensequentially incorporate desired amino acid residues utilizing generalprinciples of solid phase synthesis.

While synthesis has been described primarily with reference to solidphase Fmoc chemistry, it is to be understood that other chemistries andsynthetic methods may be employed to make the cyclic peptides of theinvention, such as by way of example and not limitation, methodsemploying Boc chemistry, solution chemistry, and other chemistries andsynthetic methods.

6.0 Formulations

Depending on the desired route of administration, the formulation of acomposition including one or more cyclic peptides of the presentinvention may be varied. Thus the formulation may be suitable forsubcutaneous injection, or intravenous injection, for topicalapplications, for ocular applications, for nasal spray applications, forinhalation applications, for other transdermal applications and thelike.

6.1 Salt Form of Cyclic Peptides of the Present Invention

The cyclic peptides of the present invention may be in the form of anypharmaceutically acceptable salt. The term “pharmaceutically acceptablesalts” refers to salts prepared from pharmaceutically acceptablenon-toxic bases or acids including inorganic or organic bases andinorganic or organic acids. Salts derived from inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic salts, manganous, potassium, sodium, zinc, and thelike. Particularly preferred are the ammonium, calcium, lithium,magnesium, potassium, and sodium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like.

When the cyclic peptide of the present invention is basic, acid additionsalts may be prepared from pharmaceutically acceptable non-toxic acids,including inorganic and organic acids. Such acids include acetic,benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric,ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonicacid, trifluoroacetic acid, and the like. Acid addition salts of thepeptides of the present invention are prepared in a suitable solventfrom the peptide and an excess of an acid, such as hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, citric,tartaric, maleic, succinic or methanesulfonic acid. The acetate,ammonium acetate and trifluoracetic acid salt forms are especiallyuseful. Where the peptides of the present invention include an acidicmoiety, suitable pharmaceutically acceptable salts may include alkalimetal salts, such as sodium or potassium salts, or alkaline earth metalsalts, such as calcium or magnesium salts.

6.2 Pharmaceutical Compositions

The invention provides a pharmaceutical composition that includes acyclic peptide of the present invention and a pharmaceuticallyacceptable carrier. The carrier may be a liquid formulation, and ispreferably a buffered, isotonic, aqueous solution. Pharmaceuticallyacceptable carriers also include excipients, such as diluents, carriersand the like, and additives, such as stabilizing agents, preservatives,solubilizing agents, buffers and the like, as hereafter described.

The cyclic peptide compositions of the present invention may beformulated or compounded into pharmaceutical compositions that includeat least one cyclic peptide of the present invention together with oneor more pharmaceutically acceptable carriers, including excipients, suchas diluents, carriers and the like, and additives, such as stabilizingagents, preservatives, solubilizing agents, buffers and the like, as maybe desired. Formulation excipients may include polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, manniton,sodium chloride and sodium citrate. For injection or other liquidadministration formulations, water containing at least one or morebuffering constituents is preferred, and stabilizing agents,preservatives and solubilizing agents may also be employed. For solidadministration formulations, any of a variety of thickening, filler,bulking and carrier additives may be employed, such as starches, sugars,fatty acids and the like. For topical administration formulations, anyof a variety of creams, ointments, gels, lotions and the like may beemployed. For most pharmaceutical formulations, non-active ingredientswill constitute the greater part, by weight or volume, of thepreparation. For pharmaceutical formulations, it is also contemplatedthat any of a variety of measured-release, slow-release orsustained-release formulations and additives may be employed, so thatthe dosage may be formulated so as to effect delivery of a peptide ofthe present invention over a period of time.

In general, the actual quantity of cyclic peptides of the presentinvention administered to a patient will vary between fairly wide rangesdepending on the mode of administration, the formulation used, and theresponse desired.

In practical use, the cyclic peptides of the invention can be combinedas the active ingredient in an admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, for example, oral, parenteral(including intravenous), urethral, vaginal, nasal, buccal, sublingual,or the like. In preparing the compositions for oral dosage form, any ofthe usual pharmaceutical media may be employed, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents and the like in the case of oral liquid preparationssuch as, for example, suspensions, elixirs and solutions; or carrierssuch as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations such as, for example,powders, hard and soft capsules and tablets.

Because of their ease of administration, tablets and capsules representan advantageous oral dosage unit form. If desired, tablets may be coatedby standard aqueous or nonaqueous techniques. The amount of activepeptide in such therapeutically useful compositions is such that aneffective dosage will be obtained. In another advantageous dosage unitform, sublingual constructs may be employed, such as sheets, wafers,tablets or the like.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch or alginic acid; a lubricant such as magnesium stearate;and a sweetening agent such as sucrose, lactose or saccharin. When adosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier such as fatty oil.

Various other materials may be utilized as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Cyclic peptides may also be administered parenterally. Solutions orsuspensions of these active peptides can be prepared in water suitablymixed with a surfactant such as hydroxy-propylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof in oils. These preparations may optionally contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that it may be administered by syringe. The form must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, a polyol, for example glycerol,propylene glycol or liquid polyethylene glycol, suitable mixturesthereof, and vegetable oils.

The cyclic peptides of the present invention may be therapeuticallyapplied by means of nasal administration. By “nasal administration” ismeant any form of intranasal administration of any of the cyclicpeptides of the present invention. The peptides may be in an aqueoussolution, such as a solution including saline, citrate or other commonexcipients or preservatives. The peptides may also be in a dry or powderformulation.

The cyclic peptides of the present invention may be formulated with anyof a variety of agents that increase effective nasal absorption ofdrugs, including peptide drugs. These agents should increase nasalabsorption without unacceptable damage to the mucosal membrane. U.S.Pat. Nos. 5,693,608, 5,977,070 and 5,908,825, among others, teach anumber of pharmaceutical compositions that may be employed, includingabsorption enhancers, and the teachings of each of the foregoing, andall references and patents cited therein, are incorporated by reference.

If in an aqueous solution, the cyclic peptides may be appropriatelybuffered by means of saline, acetate, phosphate, citrate, acetate orother buffering agents, which may be at any physiologically acceptablepH, generally from about pH 4 to about pH 7. A combination of bufferingagents may also be employed, such as phosphate buffered saline, a salineand acetate buffer, and the like. In the case of saline, a 0.9% salinesolution may be employed. In the case of acetate, phosphate, citrate,and the like, a 50 mM solution may be employed. In addition to bufferingagents, a suitable preservative may be employed, to prevent or limitbacteria and other microbial growth. One such preservative that may beemployed is 0.05% benzalkonium chloride.

In an alternative embodiment, cyclic peptides of the present inventionmay be administered directly into the lung. Intrapulmonaryadministration may be performed by means of a metered dose inhaler, adevice allowing self-administration of a metered bolus of a peptide ofthe present invention when actuated by a patient during inspiration. Inone aspect of this embodiment, the cyclic peptide may be in a dried andparticulate form, for example particles between about 0.5 and 6.0 μm,such that the particles have sufficient mass to settle on the lungsurface, and not be exhaled, but are small enough that they are notdeposited on surfaces of the air passages prior to reaching the lung.Any of a variety of different techniques may be used to make dry powdermicroparticles, including but not limited to micro-milling, spray dryingand a quick freeze aerosol followed by lyophilization. Withmicroparticles, the peptides may be deposited to the deep lung, therebyproviding quick and efficient absorption into the bloodstream. Further,with such approach penetration enhancers are not required, as issometimes the case in transdermal, nasal or oral mucosal deliveryroutes. Any of a variety of inhalers can be employed, includingpropellant-based aerosols, nebulizers, single dose dry powder inhalersand multidose dry powder inhalers. Common devices in current use includemetered dose inhalers, which are used to deliver medications for thetreatment of asthma, chronic obstructive pulmonary disease and the like.Preferred devices include dry powder inhalers, designed to form a cloudor aerosol of fine powder with a particle size that is always less thanabout 6.0 μm.

Microparticle size, including mean size distribution, may be controlledby means of the method of making. For micro-milling, the size of themilling head, speed of the rotor, time of processing and the likecontrol the microparticle size. For spray drying, the nozzle size, flowrate, dryer heat and the like control the microparticle size. For makingby means of quick freeze aerosol followed by lyophilization, the nozzlesize, flow rate, concentration of aerosoled solution and the likecontrol the microparticle size. These parameters and others may beemployed to control the microparticle size.

The cyclic peptides of the present invention may be therapeuticallyadministered by means of an injection of a sustained releaseformulation. In one embodiment, a cyclic peptide of the presentinvention is formulated for a deep intramuscular injection, such as inthe gluteal or deltoid muscle, of a formulation with a polyethyleneglycol, such as polyethylene glycol 3350, and optionally one or moreadditional excipients and preservatives, including but not limited toexcipients such as salts, polysorbate 80, sodium hydroxide orhydrochloric acid to adjust pH, and the like. In another embodiment acyclic peptide of the present invention is formulated with a poly(orthoester), which may be an auto-catalyzed poly(ortho ester) with any of avariable percentage of lactic acid in the polymeric backbone, andoptionally one or more additional excipients. In one embodiment poly(D,L-lactide-co-glycolide) polymer is employed. In general, any of anumber of injectable and bioerodible polymers, which are preferably alsoadhesive polymers, may be employed in a sustained release injectableformulation. Alternatively other sustained release formulations may beemployed, including formulations permitting subcutaneous injection,which other formulations may include one or more of nano/microspheres(such as compositions including PLGA polymers), liposomes, emulsions(such as water-in-oil emulsions), gels, insoluble salts or suspensionsin oil. The formulation may be such that an injection is required on adaily, weekly, monthly or other periodic basis, depending on theconcentration and amount of cyclic peptide, the sustained release rateof the materials employed, and other factors known to those of skill inthe art.

6.3 Oral Formulations of Peptides of the Present Invention

In one aspect, the peptides of the present invention are formulated fororal delivery. The peptide is preferably formulated and made such thatit is encased in an enteric protectant, more preferably such that it isnot released until the tablet or capsule has transited the stomach, andoptionally has further transited a portion of the small intestine. Inthe context of this application it will be understood that the termenteric coating or material refers to a coating or material that willpass through the stomach essentially intact but will rapidlydisintegrate in the small intestine to release the active drugsubstance. One enteric coating solution that may be used includescellulose acetate phthalate, and optionally other ingredients such asammonium hydroxide, triacetin, ethyl alcohol, methylene blue, andpurified water. Cellulose acetate phthalate is a polymer that has beenused in the pharmaceutical industry for enterically coating individualdosage forms such as tablets and capsules, and is not soluble in waterat a pH of less than about 5.8. Enteric coatings including celluloseacetate phthalate provide protection against the acidic environment ofthe stomach, but begin to dissolve in environment of the duodenum (pH ofabout 6-6.5), and are completely dissolved by the time the dosage formreaches the ileum (pH of about 7-8). In addition to cellulose acetatephthalate, other enteric coating materials are known and may be usedwith peptides of the present invention, including without limitationhydroxypropylmethylethylcellulose succinate,hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, andmethacrylic acid-methyl methacrylate copolymer. The enteric coatingemployed promotes dissolution of the dosage form primarily at a siteoutside the stomach, and may be selected such that the enteric coatingdissolves at a pH of approximately at least 6.0, more preferable at a pHof from about 6.0 to about 8.0. In one preferred aspect, the entericcoating dissolves and breaks down in the proximity of the ileum.

Any of a variety of permeation enhancers may be employed, to increaseuptake in the intestines upon dissolution of the enteric coating. In oneaspect, permeation enhancers increase either paracellular ortranscellular transport systems. An increase in paracellular transportcan be achieved by opening the tight junctions of the cells; an increasein transcellular transport can be achieved by increasing the fluidity ofthe cell membrane. Representative, non-limiting examples of suchpermeation enhancers include calcium chelators, bile salts (such assodium cholate), and fatty acids. The peptides of the present inventionmay be in an enteric-coated individual dosage form that includes a fattyacid, such as for example oleate, palmitate, stearate, sodium caprate,or conjugated linoleic acid, in an enteric-coated capsule, to increaseparacellular transport.

In one aspect, the individual dosage form, such as a tablet or capsule,optionally further includes common pharmaceutical binders such aspovidone, diluents, glidants, fillers such as microcrystallinecellulose, lubricants such as magnesium stearate, disintegrants such ascroscarmellose sodium, preservatives, colorants and the like in theirusual known sizes and amounts. In some embodiments, peptides orpolypeptides that act as substrates for intestinal proteases are furtheradded.

6.4 Ophthalmic Formulations

In one embodiment, ocular diseases, indications, conditions andsyndromes, such as for example either dry eye disease or uveitis, may betreated with an ophthalmic dosage form including one or more of thepeptides of the present invention. The ophthalmic dosage form mayinclude one or more active ingredients in addition to one or more of thepeptides of the present invention, such as for example artificial tearcomponents, topical corticosteroids, non-steroidal anti-inflammatorydrugs, or calcineurin inhibitors such as cyclosporine-A(Restasis®—Allergan). In a related embodiment, one or additionalcompounds may be given separately from one or more of the peptides ofthe present invention, such as separate administration of an ophthalmicdosage form including an artificial tear component, a topicalcorticosteroid, a non-steroidal anti-inflammatory drugs, a calcineurininhibitor such a cyclosporine-A, or a combination of any of theforegoing.

Ophthalmic solutions are preferably maintained in a pH range betweenabout pH 3.5 to 9.0, and preferably about pH 6.5 and pH 7.2, with asuitable buffer. The pH may be adjusted by any known means, such as byuse of HCl or NaOH. Buffers may include acetate, boric acid, sodiumborate, potassium citrate, citric acid, sodium bicarbonate, TRIS,various mixed phosphate buffers (such as combinations of Na₂HPO₄,NaH₂PO₄ and KH₂PO₄) and mixtures thereof. Generally, buffers will beused in amounts ranging from about 0.05% to 2.5% (w/v), and preferablyfrom about 0.1% to 1.5% percent; buffers should be as close tophysiological ion concentrations as possible to minimize potentialirritation but still maintain drug product pH over the shelf life of theproduct.

The ophthalmic solutions employed in the present invention may be madefrom purified water, and in one aspect preferably from a physiologicalsaline solution. Additional tonicity enhancing agents may be employed,including either ionic or non-ionic tonicity enhancing agents, or both.Ionic tonicity enhancing agents include alkali metal or earth metalhalides, such as CaCl₂, KBr, KCl, LiCl, NaI, NaBr, NaCl, Na₂SO₄ or boricacid. Non-ionic tonicity enhancing agents include urea, glycerol,sorbitol, mannitol, propylene glycol, or dextrose. The aqueous solutionsof the present invention are typically adjusted with tonicity agents toapproximate an osmotic pressure equivalent to a 0.9% (w/v) solution ofsodium chloride or a 2.5% solution of glycerol. However, tonicity rangesequivalent to between 0.7% and 1.5% NaCl are generally considered to beacceptable.

The solutions can also contain conventional, pharmaceutically acceptablepreservatives, stabilizers, cosolvents and/or penetration enhancers aswell as viscoelastic substances included in artificial tearpreparations. Pharmaceutically acceptable preservatives includequaternary ammonium compounds such as benzalkonium chloride, benzoxoniumchloride or the like; alkyl-mercury salts of thiosalicylic acid such asthiomersal, phenylmercuric nitrate, phenylmercuric acetate orphenylmercuric borate; sodium perborate; sodium chlorite; parabens, suchasmethylparaben or propylparaben; alcohols such as chlorobutanol, benzylalcohol or phenyl ethanol; guanidine derivatives such as chlorohexidineor polyhexamethylene biguanide; sorbic acid; boric acid; or peroxideforming preservatives, or combinations of two or more of the foregoing.Pharmaceutically acceptable antioxidants and chelating agents may beused including various sulphites (such as sodium metabisulphite, sodiumthiosulphate, sodium bisulfite, or sodium sulfite), α-tocopherol,ascorbic acid, acetylcysteine, 8-hydroxyquinolome, antipyrine, butylatedhydroxyanisole or butylated hydroxytoluene, EDTA, and others. Cosolventssuch as alcohols and others may also be used. Various substances canalso be used to enhance formulation stability, such as cyclodextrins.

Penetration enhancers may be employed in ophthalmic solutions, includingcompounds such as surfactants, certain organic solvents such asdimethylsulphoxide and other sulphoxides, dimethylacetamide andpyrrolidine, certain amides of heterocyclic amines, glycols (e.g.propylene glycol), propylene carbonate, oleic acid, alkylamines andderivatives, various cationic, anionic and nonionic surfactants,amphoteric surfactants and the like. Additional penetration enhancersthat may be employed include cetylpyridinium chloride, ionophores suchas lasalocid, benzalkonium chloride, polysorbates such as polysorbate 20(Tween® 20), parabens, saponins, various polyoxyethylene ether compoundssuch as Brij® 35, Brij® 78 or Brij® 98, ethylenediaminetetraacetic acid(EDTA), bile salts, and bile acids (such as sodium cholate, sodiumtaurocholate, sodium glycodeoxycholate, sodium taurodeoxycholate,taurocholic acid, chenodeoxycholic acid and ursodeoxycholic acid),capric acid, azone, fucidic acid, hexamethylene lauramide, saponins,hexamethylene octanamide, and decylmethyl sulfoxide. Ion pairingformulations utilizing charged excipients or counter ions toshield/neutralize charged groups on drug molecules may also be employedto lower the lipophilicity of the compound to increase cornealpenetration. These formulations include but are not limited to ions suchas sorbic acid, boric acid and maleic acid, among other charged ionpairing agents.

Viscosity enhancers or lubricants may be employed as necessary orappropriate. In one aspect, the viscosity enhancer includes a watersoluble polymer, such as polyols, including polyvinyl alcohol, apolyethylene glycol, or combinations of water soluble polymers. In oneaspect, polyethylene glycol 300 or 400 is employed. The contents ofwater soluble polymer may be between about 0.25% and about 4.0% (w/v).Thus an ophthalmic solution can include, by way of example, 1% ofpolyvinyl alcohol, 1% of polyethylene glycol 300 or 400, or both. Otherpolyols may be employed, including glycerol, glycerin, polysorbate 80,propylene glycol, ethylene glycol, povidone, and polyvinylpyrrolidone.Other lubricants, sometimes also called tear substitutes, may also beemployed, including cellulose derivatives such hydroxypropyl methylcellulose, carboxymethyl cellulose sodium, hydroxypropyl cellulose,hydroxyethyl cellulose, and methyl cellulose; dextrans such as dextran70; water soluble proteins such as gelatin; carbomers such as carbomer934P, carbomer 941, carbomer 940 and carbomer 974P; and gums such asHP-guar, xanthan gum or combinations thereof. Other viscosity enhancersthat can be employed include polysaccharide compounds, such as sulfatedor non-sulfated glycosaminoglycan compounds. In one aspect, thepolysaccharide compound is a non-sulfated glycosaminoglycan such ashyaluronic acid or a pharmaceutically acceptable salt thereof, such assodium hyaluronate. Any commercially available molecular weight range ofhyaluronic acid or salts thereof may be employed. From about 0.05% toabout 0.4% (w/v) of hyaluronic acid or a salt thereof may be employed inan ophthalmic solution. In another aspect, the polysaccharide compoundis a non-sulfated glycosaminoglycan such as dextran. In yet anotheraspect, the polysaccharide is a sulfated glycosaminoglycan such aschondroitin sulfate.

Semi-solid formulations may be employed for ophthalmic delivery toincrease corneal residence times of drug molecules. Ointments containingpolyethylene glycols, lanolin alcohols, ozokerite, ceresin,microcrystalline wax, surfactants, preservatives, sorbitan monolaurate,white petrolatum and light liquid petrolatum (mineral oil) or otherpetrolatum like bases may be used. Aqueous or non-aqueous suspensionsmay also be used. For hydrophilic peptides, suspensions usingpharmaceutically acceptable oils or petrolatum may be used. Suspensionsmay contain microspheres or microparticulates, nanoparticulates,mucoadhesive particles, viscosity increasing agents, surfactants andother agents. Mucoadhesive compounds include synthetic polymers, such aspolyacrylic acid and polycarbophil; biopolymers such as hyaluronic acidor sodium carboxy methylcellulose (CMC); polyanionic polymers such aspolyacrylic acid (PAA); polyacrylic acids such as Carbopol® 934P,polycarbophil, and CMC or PAA with Pluronic® polyoxalkylene ethers; orpolycationic polymers such as chitosan. Emulsions (oil in water or waterin oil), including microemulsions, may also be employed that arecomposed of pharmaceutically acceptable oils together with one or moreof viscosity increasing agents, preservatives, cosolvents, surfactantsand other agents. Pharmaceutically acceptable oils include mineral oilsand organic oils, including oils comprising medium chain or long chainsaturated or unsaturated fatty acids or esters thereof. Pharmaceuticallyacceptable oils thus include any of a range of medium chaintriglycerides, as well as oils such as almond oil, castor oil,cottonseed oil, glycerin (glycerol), peanut oil, mineral oil,polyethylene glycol, poppyseed oil, propylene glycol, safflower oil,sesame oil, soybean oil, olive oil and vegetable oil. A surfactant suchas a polyoxyethylene alkyl ether, polyoxyl castor oil, tyloxapol, alkylaryl ether sulfonate, lecithin, sorbitan esters, glyceryl monostearate,cetyl alcohol, octoxynol-9, nonoxynol-9, polyoxyethylene stearates,polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 60and 80 or others may also be employed. Aqueous gels, often comprised ofpolymers such as polyvinyl alcohol (PVA), polyacrylamide, poloxamer,hydroxypropyl methylcellulose (HPMC), carbomer, polymethylvinylethermaleic anhydride, and hydroxypropyl ethylcellulose may also be used.Hydrogels containing swellable, water insoluble polymers may be utilizedcontaining polymers such as poly(acrylic acid), poly(acrylic acids),poly(acrylamide), and ethylene maleic anhydride, and chemically orthermally-treated gelatins. Ocular inserts, liposomes, discomes,niosomes, dedrimers, nanosuspensions, nanoparticles and microparticlesmay also be used to provide a controlled release of the drug. Liposomesand other controlled release agents may be positively charged toincrease residence times through ionic interactions with the negativelycharged corneal surface. Nanoparticles may be composed of biodegradablepolymers such as polyactides (PLAs), polycyano acrylates, poly(D,L-lactides), and natural polymers such as chitosan, gelatin, sodiumalginate, albumin and others.

6.5 Routes of Administration of Formulations

If a formulation including one or more peptides of the present inventionis administered by injection, the injection may be intravenous,subcutaneous, intramuscular, intraperitoneal or other means known in theart. The peptides of the present invention may be formulated by anymeans known in the art, including but not limited to formulation astablets, capsules, caplets, suspensions, powders, lyophilizedpreparations, suppositories, ocular drops, skin patches, oral solubleformulations, sprays, aerosols and the like, and may be mixed andformulated with buffers, binders, excipients, stabilizers, anti-oxidantsand other agents known in the art. In general, any route ofadministration by which the peptides of invention are introduced acrossan epidermal layer of cells may be employed.

Administration means may thus include formulations for administrationthrough mucous membranes, buccal administration, oral administration,dermal administration, inhalation administration, nasal administration,urethral administration, vaginal administration, and the like.

6.6 Therapeutically Effective Amount

In general, the actual quantity of cyclic peptide of the presentinvention administered to a patient will vary between fairly wide rangesdepending upon the mode of administration, the formulation used, and theresponse desired. The dosage for treatment is administration, by any ofthe foregoing means or any other means known in the art, of an amountsufficient to bring about the desired therapeutic effect. Thus atherapeutically effective amount includes an amount of a peptide orpharmaceutical composition of the present invention that is sufficientto therapeutically alleviate sexual dysfunction in a patient, or toprevent or delay onset or recurrence of the sexual dysfunction.

In general, the cyclic peptides of the present invention are highlyactive. For example, the cyclic peptide can be systemically administeredat about 0.1, 0.5, 1, 5, 50, 100, 500, 1000 or 5000 μg/kg body weight,depending on the specific peptide selected, the desired therapeuticresponse, the route of administration, the formulation and other factorsknown to those of skill in the art.

7.0 Peptides of the Present Invention

In one aspect, the invention provides a cyclic peptide which contains acore sequence derived from His-Phe-Arg within the cyclic portion, butnot including Trp within the core portion, and where Trp, or aderivative or mimetic thereof (defined as an amino acid residue with aside chain including at least one aryl or heteroaryl, including but notlimited to Nal 1 or Nal 2), is the amino acid residue immediatelyoutside the cyclic portion on the C-terminus side. Thus the sequence isderived from His-Phe-Arg-Xaa⁶-Trp, where Xaa⁶ is an amino acid whereinthe side chain thereof forms a cyclic bridge with either the side chainof another amino acid or the N-terminus group of the peptide.

The core sequence derived from His-Phe-Arg-Xaa⁶-Trp may include a numberof substitutions. The His position may be His, or may be a substitutedor unsubstituted Pro or an amino acid with a side chain including atleast one primary amine, secondary amine, alkyl, cycloalkyl,cycloheteroalkyl, aryl, heteroaryl, alcohol, ether, sulfide, sulfone,sufoxide, carbomyl or carboxyl. Substituted Pro includes, but is notlimited to, amino acids such as Hyp, Hyp(Bzl), Pro(4R-Bzl) orPro(4R—NH₂). The Phe position may be Phe, but is most typicallysubstituted or unsubstituted D-Phe, D-Nal 1, D-Nal 2 or an amino acidwith a side chain including pyridyl. The Arg position may be Arg, Lys,Orn, Dab or Dap, or a substituted or unsubstituted Pro, or Cit, or maybe an amino acid with a side chain including at least one primary amine,secondary amine, guanidine, urea, alkyl, cycloalkyl, cycloheteroalkyl,aryl, heteroaryl, or ether. Xaa⁶ may be an amino acid with a side chaininclude a primary amine, such as Lys, Orn, Dab, Dap, an amino acid witha carboxyl group, such as Asp, Glu or hGlu, or an amino acid with adisulfide group, such as Cys or Pen, all depending on the nature of thecyclic bridge. The Trp position may be an amino acid with a side chainincluding at least one substituted or unsubstituted aryl or heteroaryl,such as Trp, Nal 1 or Nal 2.

The invention thus provides a cyclic peptide of formula (VII):

Z-Xaa ¹-Xaa ²-Xaa ³-Xaa ⁴-Xaa ⁵-Xaa ⁶-Xaa ⁷-Y  (VII)

or a pharmaceutically acceptable salt thereof, wherein:

Z is H or an N-terminal group;

Xaa¹ is optionally present, and if present is from one to three L- orD-isomer amino acid residues;

Xaa² and Xaa⁶ are L- or D-isomer amino acids wherein the side chainsthereof comprise a cyclic bridge;

Xaa³ is L- or D-Pro, optionally substituted with hydroxyl, halogen,sulfonamide, alkyl, —O-alkyl, aryl, alkyl-aryl, alkyl-O-aryl,alkyl-O-alkyl-aryl, or —O-aryl, or Xaa³ is an L- or D-isomer of an aminoacid with a side chain including at least one primary amine, secondaryamine, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, ether,sulfide, or carboxyl;

Xaa⁴ is an L- or D-isomer amino acid with a side chain including phenyl,naphthyl or pyridyl, optionally wherein the ring is substituted with oneor more substituents independently selected from halo,(C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy, (C₁-C₁₀)alkylthio,aryl, aryloxy, nitro, nitrile, sulfonamide, amino, monosubstitutedamino, disubstituted amino, hydroxy, carboxy, and alkoxy-carbonyl;

Xaa⁵ is L- or D-Pro or Xaa⁵ is an L- or D-isomer amino acid with a sidechain including at least one primary amine, secondary amine, guanidine,urea, alkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, or ether;

Xaa⁷ is optionally present, and if present is from one to three L- orD-isomer amino acid residues; and

Y is a C-terminal group.

In one aspect, Xaa⁴ may be D-Phe, optionally substituted with one ormore substituents independently selected from halo, (C₁-C₁₀)alkyl-halo,(C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy, (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro,nitrile, sulfonamide, amino, monosubstituted amino, disubstituted amino,hydroxy, carboxy, and alkoxy-carbonyl.

In another aspect, one of Xaa² and Xaa⁶ may be an L- or D-isomer of Asp,hGlu or Glu and the other of Xaa² and Xaa⁶ is an L- or D-isomer of Lys,Orn, Dab or Dap. In an alternative aspect, each of Xaa² and Xaa⁶ may beCys, D-Cys, Pen or D-Pen.

In another aspect, Xaa¹ may be an amino acid with a side chain includinga linear or branched alkyl, cycloalkyl, cycloheteroalkyl, aryl orheteroaryl.

In another aspect, Xaa⁷ may be an amino acid with a side chain includingat least one aryl or heteroaryl, optionally substituted with one or morering substituents, and when one or more substituents are present, arethe same or different and independently hydroxyl, halogen, sulfonamide,alkyl, —O-alkyl, aryl, or —O-aryl.

In another aspect, the N-terminal group may be a C₁ to C₁₇ acyl group,wherein the C₁ to C₁₇ comprises a linear or branched alkyl, cycloalkyl,alkylcycloalkyl, aryl or alkylaryl, a linear or branched C₁ to C₁₇alkyl, aryl, heteroaryl, alkene, alkenyl, or aralkyl chain or anN-acylated linear or branched C₁ to C₁₇ alkyl, aryl, heteroaryl, alkene,alkenyl, or aralkyl chain.

In another aspect, Y may be a hydroxyl, an amide, or an amidesubstituted with one or two linear or branched C₁ to C₁₇ alkyl,cycloalkyl, aryl, alkyl cycloalkyl, aralkyl, heteroaryl, alkene,alkenyl, or aralkyl chains.

The invention thus provides in another aspect a cyclic peptide offormula (VII) defined as above, but wherein

Xaa⁴ is D-Phe, optionally substituted with one or more substituentsindependently selected from halo, (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl,(C₁-C₁₀)alkoxy, (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro, nitrile,sulfonamide, amino, monosubstituted amino, disubstituted amino, hydroxy,carboxy, and alkoxy-carbonyl;

Xaa⁵ is an L- or D-isomer of Arg, Lys, Orn, Dab or Dap; and

Xaa⁷ is an L- or D-isomer of Trp, Nal 1 or Nal 2.

In the foregoing, in one aspect Xaa³ may be an L- or D-isomer of His,and in another aspect Z may be a C₁ to C₁₇ acyl group and Xaa¹ may be anL- or D-isomer of Nle.

In the foregoing, and in formula (I), substituted Pro may be, forexample, Hyp, Hyp(Bzl), Pro(4-Bzl), and Pro(4-NH₂).

The peptides encompassed within formulas (I) through (VII) contain oneor more asymmetric elements such as stereogenic centers, stereogenicaxes and the like, so that the peptides encompassed within formula (I)can exist in different stereoisomeric forms. For both specific andgenerically described peptides, including the peptides encompassedwithin formulas (I) through (VI), all forms of isomers at all chiral orother isomeric centers, including enantiomers and diastereomers, areintended to be covered herein. The peptides of the invention eachinclude multiple chiral centers, and may be used as a racemic mixture oran enantiomerically enriched mixture, in addition to use of the peptidesof the invention in enantiopure preparations. Typically, the peptides ofthe invention will be synthesized with the use of chirally purereagents, such as specified L- or D-amino acids, using reagents,conditions and methods such that enantiomeric purity is maintained, butit is possible and contemplated that racemic mixtures may be made. Suchracemic mixtures may optionally be separated using well-known techniquesand an individual enantiomer may be used alone. In cases and underspecific conditions of temperature, solvents and pH wherein peptides mayexist in tautomeric forms, each tautomeric form is contemplated as beingincluded within this invention whether existing in equilibrium orpredominantly in one form. Thus a single enantiomer of a peptide offormula (I), which is an optically active form, can be obtained byasymmetric synthesis, synthesis from optically pure precursors, or byresolution of the racemates.

The invention is further intended to include prodrugs of the presentpeptides, which on administration undergo chemical conversion bymetabolic processes before becoming active pharmacological peptides. Ingeneral, such prodrugs will be functional derivatives of the presentpeptides, which are readily convertible in vivo into a peptide offormula (I) through (VII). Prodrugs are any covalently bonded compounds,which release the active parent peptide drug of formula (I) through(VII) in vivo. Conventional procedures for the selection and preparationof suitable prodrug derivatives are described, for example, in “Designof Prodrugs”, ed. H. Bundgaard, Elsevier, 1985. Typical examples ofprodrugs have biologically labile protecting groups on a functionalmoiety, such as for example by esterification of hydroxyl, carboxyl oramino functions. Thus by way of example and not limitation, a prodrugincludes peptides of formula (I) wherein an ester prodrug form isemployed, such as, for example, lower alkyl esters of an R group offormula (I), such as where R is —OH, which lower alkyl esters mayinclude from 1-8 carbons in an alkyl radical or aralkyl esters whichhave 6-12 carbons in an aralkyl radical. Broadly speaking, prodrugsinclude compounds that can be oxidized, reduced, aminated, deaminated,hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated,dealkylated, acylated, deacylated, phosphorylated or dephosphorylated toproduce an active parent peptide drug of formula (I) in vivo.

The subject invention also includes peptides which are identical tothose recited in formula (I) through (VI), but for the fact that one ormore atoms depicted in formula (I) through (VI) are replaced by an atomhaving an atomic mass or mass number different from the atomic mass ormass number usually found in nature. Examples of isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen and oxygen, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸O and ¹⁷O, respectively. Peptides of the present invention andpharmaceutically acceptable salts or solvates of said compounds whichcontain the aforementioned isotopes and/or other isotopes of other atomsare within the scope of this invention. Certain isotopically-labeledcompounds of the present invention, for example those into whichradioactive isotopes such as ³H and ¹⁴C are incorporated, may have usein a variety of assays, such as in drug and/or substrate tissuedistribution assays. Substitution with heavier isotopes, such assubstitution of one or more hydrogen atoms with deuterium (²H), canprovide pharmacological advantages in some instances, includingincreased metabolic stability. Isotopically labeled peptides of formula(I) through (VI) can generally be prepared by substituting anisotopically labeled reagent for a non-isotopically labeled reagent.

8.0 Tests and Assays Employed in Evaluation of the Peptides of thePresent Invention

The melanocortin receptor-specific peptides of the present invention ofthis invention may be tested by a variety of assay systems and animalmodels to determine binding, functional status and efficacy.

8.1 Competitive Inhibition Assay Using [I¹²⁵]-NDP-α-MSH

A competitive inhibition binding assay was performed using membranehomogenates prepared from HEK-293 cells that express recombinant hMCR-laor hMCR-4 (in each instance where the h prefix refers to human), oralternatively membrane homogenates from B16-F10 mouse melanoma cellscontaining endogenous murine MCR-1. In the examples that follow, allMCR-1 and MCR-4 values are for human recombinant receptors, unlessotherwise noted. Assays were performed in 96 well polypropyleneround-bottom plates (VWR catalog number 12777-030). Membrane homogenateswere incubated with 0.1 nM [I¹²⁵]-NDP-α-MSH (Perkin Elmer) andincreasing concentrations of test peptides of the present invention inbuffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serumalbumin. After incubation for 90 minutes at 37° C., the assay mixturewas filtered onto GF/B Unifilter plates (Perkin-Elmer catalog number6005177) and washed with 3 mL of ice-cold buffer per well. Filters wereair dried and 35 μL of scintillation cocktail added to each well. Plateswere counted in a Microbeta counter for bound radioactivity.Non-specific binding was measured by inhibition of binding of[I¹²⁵]-NDP-α-MSH in the presence of 1 μM NDP-α-MSH. Maximal specificbinding (100%) was defined as the difference in radioactivity (cpm)bound to cell membranes in the absence and presence of 1 μM NDP-α-MSH.Each assay was conducted in duplicate and the actual mean values aredescribed, with results less than 0% reported as 0%. Ki values forpeptides of the present invention were determined using Graph-Pad Prism®curve-fitting software.

8.2 Assay for Agonist Activity

Accumulation of intracellular cAMP was examined as a measure of theability of the peptides of the present invention to elicit a functionalresponse in a human melanoma cell line, HBL, that express hMCR-1 (seeKang, L., et al., “A selective small molecule agonist of melanocortin-1receptor inhibits lipopolysaccharide-induced cytokine accumulation andleukocyte infiltration in mice,” J. Leuk. Biol. 80:897-904 (2006)) orHEK-293 cells that express hMCR-4. Confluent HBL cells that expresshMCR-1 or HEK-293 cells that express recombinant hMCR-4 were detachedfrom culture plates by incubation in enzyme-free cell dissociationbuffer. Dispersed cells were suspended in Earle's Balanced Salt Solutioncontaining 10 mM HEPES (pH 7.5), 1 mM MgCl₂, 1 mM glutamine, 0.5%albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), aphosphodiesterase inhibitor. The cells were plated in 96-well plates ata density of 0.4×10⁵ cells per well for HBL cells and 0.5×10⁵ cells perwell for HEK-293 cells and pre-incubated for 10 minutes. Cells wereexposed for 15 minutes at 37° C. to peptides of the present inventiondissolved in DMSO (final DMSO concentration of 1%) at a concentrationrange of 0.05-5000 nM in a total assay volume of 200 μL. NDP-α-MSH wasused as the reference agonist. cAMP levels were determined by an HTRF®cAMP cell-based assay system from Cisbio Bioassays utilizingcryptate-labeled anti-cAMP and d2-labeled cAMP, with plates read on aPerkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis wasperformed by nonlinear regression analysis with Graph-Pad Prism®software. Maximum efficacy (E_(max)) values were determined for eachtest peptide of the present invention, compared to that achieved by thereference melanocortin agonist NDP-α-MSH.

9.0 Examples

Peptides of the following structures were synthesized and averaged MCR-1and MCR-4 Ki values were determined as indicated. All Ki values weredetermined using [I¹²⁵]-NDP-α-MSH. All results are expressed in nMexcept for E_(max) values, which are percentage values. Peptides withthe captioned primary sequence were synthesized and purified asdescribed in Section 5 above, with the resulting peptide having thestructure depicted. After synthesis and purification, each peptide wastested as described in Section 8 above, with the results as shown.

9.1

Assay Result MC-4 Ki (average) 45 MC-1 Ki (average)  0.01 MC-1 EC₅₀(average; cAMP HBL)  0.007 MC-1 E_(max) (average; cAMP HBL) 97% 9.2

Assay Result MC-4 Ki (average) 110 MC-1 Ki (average)  0.012 MC-1 EC₅₀(average; cAMP HBL)  0.006 MC-1 E_(max) (average; cAMP HBL)  95% 9.3

Assay Result MC-4 Ki (average) 358 MC-1 Ki (average)  0.04 MC-1 EC₅₀(average; cAMP HBL)  0.008 MC-1 E_(max) (average; cAMP HBL)  91% 9.4

Assay Result MC-4 Ki (average) 1325 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.195 MC-1 E_(max) (average; cAMP HBL)  88% 9.5

Assay Result MC-4 Ki (average) 540 MC-1 Ki (average)  0.35 MC-1 EC₅₀(average; cAMP HBL)  0.025 MC-1 E_(max) (average; cAMP HBL)  87% 9.6

Assay Result MC-4 Ki (average) 295 MC-1 Ki (average)  0.07 MC-1 EC₅₀(average; cAMP HBL)  0.014 MC-1 E_(max) (average; cAMP HBL)  91% 9.7

Assay Result MC-4 Ki (average) 33 MC-1 Ki (average)  0.55 MC-1 EC₅₀(average; cAMP HBL)  0.025 MC-1 E_(max) (average; cAMP HBL) 93% 9.8

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.052 MC-1 E_(max) (average; cAMP HBL)   88% 9.9

Assay Result MC-4 Ki (average)  2 MC-1 Ki (average)  0.1 MC-1 EC₅₀(average; cAMP HBL)  0.008 MC-1 E_(max) (average; cAMP HBL) 73% 9.10

Assay Result MC-4 Ki (average)  3 MC-1 Ki (average)  0.01 MC-1 EC₅₀(average; cAMP HBL)  0.004 MC-1 E_(max) (average; cAMP HBL) 83% 9.11

Assay Result MC-4 Ki (average)  0.095 MC-1 Ki (average)  0.02 MC-1 EC₅₀(average; cAMP HBL)  0.005 MC-1 E_(max) (average; cAMP HBL) 69% 9.12

Assay Result MC-4 Ki (average) 535 MC-1 Ki (average)  0.35 MC-1 EC₅₀(average; cAMP HBL)  0.015 MC-1 E_(max) (average; cAMP HBL)  75% 9.13

Assay Result MC-4 Ki (average) 510 MC-1 Ki (average)  0.195 MC-1 EC₅₀(average; cAMP HBL)  0.01 MC-1 E_(max) (average; cAMP HBL)  75% 9.14

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)  310 MC-1 EC₅₀(average; cAMP HBL)   31 MC-1 E_(max) (average; cAMP HBL)   80% 9.15

Assay Result MC-4 Ki (average) 890 MC-1 Ki (average)  0.65 MC-1 EC₅₀(average; cAMP HBL)  0.012 MC-1 E_(max) (average; cAMP HBL)  91% 9.16

Assay Result MC-4 Ki (average) 365 MC-1 Ki (average)  0.12 MC-1 EC₅₀(average; cAMP HBL)  0.005 MC-1 E_(max) (average; cAMP HBL)  89% 9.17

Assay Result MC-4 Ki (average) 110 MC-1 Ki (average)  0.025 MC-1 EC₅₀(average; cAMP HBL)  0.004 MC-1 E_(max) (average; cAMP HBL)  90% 9.18

Assay Result MC-4 Ki (average) 100 MC-1 Ki (average)  0.015 MC-1 EC₅₀(average; cAMP HBL)  0.003 MC-1 E_(max) (average; cAMP HBL)  87% 9.19

Assay Result MC-4 Ki (average) 640 MC-1 Ki (average)  3 MC-1 EC₅₀(average; cAMP HBL)  0.031 MC-1 E_(max) (average; cAMP HBL)  83% 9.20

Assay Result MC-4 Ki (average) 165 MC-1 Ki (average)  0.025 MC-1 EC₅₀(average; cAMP HBL)  0.004 MC-1 E_(max) (average; cAMP HBL)  79% 9.21

Assay Result MC-4 Ki (average) 93 MC-1 Ki (average)  0.01 MC-1 EC₅₀(average; cAMP HBL)  0.004 MC-1 E_(max) (average; cAMP HBL) 83% 9.22

Assay Result MC-4 Ki (average) 63 MC-1 Ki (average)  0.01 MC-1 EC₅₀(average; cAMP HBL)  0.004 MC-1 E_(max) (average; cAMP HBL) 85% 9.23

Assay Result MC-4 Ki (average) 205 MC-1 Ki (average)  0.04 MC-1 EC₅₀(average; cAMP HBL)  0.007 MC-1 E_(max) (average; cAMP HBL)  82% 9.24

Assay Result MC-4 Ki (average) 285 MC-1 Ki (average)  0.03 MC-1 EC₅₀(average; cAMP HBL)  0.006 MC-1 E_(max) (average; cAMP HBL)  83% 9.25

Assay Result MC-4 Ki (average) 7475 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.08 MC-1 E_(max) (average; cAMP HBL)  97% 9.26

Assay Result MC-4 Ki (average) 4700 MC-1 Ki (average)   1 MC-1 EC₅₀(average; cAMP HBL)   0.107 MC-1 E_(max) (average; cAMP HBL)  81% 9.27

Assay Result MC-4 Ki (average) 380 MC-1 Ki (average)  0.015 MC-1 EC₅₀(average; cAMP HBL)  0.009 MC-1 E_(max) (average; cAMP HBL)  92% 9.28

Assay Result MC-4 Ki (average) 680 MC-1 Ki (average)  0.03 MC-1 EC₅₀(average; cAMP HBL)  0.007 MC-1 E_(max) (average; cAMP HBL)  70% 9.29

Assay Result MC-4 Ki (average) 325 MC-1 Ki (average)  0.025 MC-1 EC₅₀(average; cAMP HBL)  0.006 MC-1 E_(max) (average; cAMP HBL)  78% 9.30

Assay Result MC-4 Ki (average)  0.75 MC-1 Ki (average)  0.005 MC-1 EC₅₀(average; cAMP HBL)  0.002 MC-1 E_(max) (average; cAMP HBL) 72% 9.31

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.095 MC-1 E_(max) (average; cAMP HBL)   79% 9.32

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   7 MC-1 EC₅₀(average; cAMP HBL)   0.32 MC-1 E_(max) (average; cAMP HBL)   85% 9.33

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)  645 MC-1 EC₅₀(average; cAMP HBL)   44 MC-1 E_(max) (average; cAMP HBL)   73% 9.34

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   9 MC-1 EC₅₀(average; cAMP HBL)   4 MC-1 E_(max) (average; cAMP HBL)   59% 9.35

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   0.8 MC-1 EC₅₀(average; cAMP HBL)   0.115 MC-1 E_(max) (average; cAMP HBL)   72% 9.36

Assay Result MC-4 Ki (average) 455 MC-1 Ki (average)  4 MC-1 EC₅₀(average; cAMP HBL)  0.21 MC-1 E_(max) (average; cAMP HBL)  86% 9.37

Assay Result MC-4 Ki (average)  20 MC-1 Ki (average)  0.014 MC-1 EC₅₀(average; cAMP HBL)  0.003 MC-1 E_(max) (average; cAMP HBL) 100% 9.38

Assay Result MC-4 Ki (average) 98 MC-1 Ki (average)  0.45 MC-1 EC₅₀(average; cAMP HBL)  0.065 MC-1 E_(max) (average; cAMP HBL) 97% 9.39

Assay Result MC-4 Ki (average) 7375 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.175 MC-1 E_(max) (average; cAMP HBL)  91% 9.40

Assay Result MC-4 Ki (average)  95 MC-1 Ki (average)  0.04 MC-1 EC₅₀(average; cAMP HBL)  0.006 MC-1 E_(max) (average; cAMP HBL) 108% 9.41

Assay Result MC-4 Ki (average)  98 MC-1 Ki (average)  0.05 MC-1 EC₅₀(average; cAMP HBL)  0.008 MC-1 E_(max) (average; cAMP HBL) 108% 9.42

Assay Result MC-4 Ki (average)  45 MC-1 Ki (average)  0.015 MC-1 EC₅₀(average; cAMP HBL)  0.002 MC-1 E_(max) (average; cAMP HBL) 109% 9.43

Assay Result MC-4 Ki (average) 860 MC-1 Ki (average)  0.065 MC-1 EC₅₀(average; cAMP HBL)  0.016 MC-1 E_(max) (average; cAMP HBL)  85% 9.44

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   7 MC-1 EC₅₀(average; cAMP HBL)   0.32 MC-1 E_(max) (average; cAMP HBL)   85% 9.45

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)  645 MC-1 EC₅₀(average; cAMP HBL)   44 MC-1 E_(max) (average; cAMP HBL)   73% 9.46

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average) 10000 MC-1 EC₅₀(average; cAMP HBL) NA MC-1 E_(max) (average; cAMP HBL)   51% 9.47

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   9 MC-1 EC₅₀(average; cAMP HBL)   4 MC-1 E_(max) (average; cAMP HBL)   59% 9.48

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   0.8 MC-1 EC₅₀(average; cAMP HBL)   0.115 MC-1 E_(max) (average; cAMP HBL)   72% 9.49

Assay Result MC-4 Ki (average) 455 MC-1 Ki (average)  4 MC-1 EC₅₀(average; cAMP HBL)  0.21 MC-1 E_(max) (average; cAMP HBL)  86% 9.50

Assay Result MC-4 Ki (average)  20 MC-1 Ki (average)  0.014 MC-1 EC₅₀(average; cAMP HBL)  0.003 MC-1 E_(max) (average; cAMP HBL) 100% 9.51

Assay Result MC-4 Ki (average) 98 MC-1 Ki (average)  0.45 MC-1 EC₅₀(average; cAMP HBL)  0.065 MC-1 E_(max) (average; cAMP HBL) 97% 9.52

Assay Result MC-4 Ki (average) 7375 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.175 MC-1 E_(max) (average; cAMP HBL)  91% 9.53

Assay Result MC-4 Ki (average)  95 MC-1 Ki (average)  0.04 MC-1 EC₅₀(average; cAMP HBL)  0.006 MC-1 E_(max) (average; cAMP HBL) 108% 9.54

Assay Result MC-4 Ki (average)  98 MC-1 Ki (average)  0.05 MC-1 EC₅₀(average; cAMP HBL)  0.008 MC-1 E_(max) (average; cAMP HBL) 108% 9.55

Assay Result MC-4 Ki (average)  45 MC-1 Ki (average)  0.015 MC-1 EC₅₀(average; cAMP HBL)  0.002 MC-1 E_(max) (average; cAMP HBL) 109% 9.56

Assay Result MC-4 Ki (average) 3625 MC-1 Ki (average)   4 MC-1 EC₅₀(average; cAMP HBL)   0.5 MC-1 E_(max) (average; cAMP HBL)  102% 9.57

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   30 MC-1 EC₅₀(average; cAMP HBL)   1 MC-1 E_(max) (average; cAMP HBL)   88% 9.58

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   35 MC-1 EC₅₀(average; cAMP HBL)   2 MC-1 E_(max) (average; cAMP HBL)   86% 9.59

Assay Result MC-4 Ki (average) 1235 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.1 MC-1 E_(max) (average; cAMP HBL)  100% 9.60

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   18 MC-1 EC₅₀(average; cAMP HBL)   0.8 MC-1 E_(max) (average; cAMP HBL)   95% 9.61

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   20 MC-1 EC₅₀(average; cAMP HBL)   1 MC-1 E_(max) (average; cAMP HBL)  102% 9.62

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   40 MC-1 EC₅₀(average; cAMP HBL)   1 MC-1 E_(max) (average; cAMP HBL)   97% 9.63

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   3 MC-1 EC₅₀(average; cAMP HBL)   0.076 MC-1 E_(max) (average; cAMP HBL)   96% 9.64

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)   2 MC-1 EC₅₀(average; cAMP HBL)   0.12 MC-1 E_(max) (average; cAMP HBL)   99% 9.65

Assay Result MC-4 Ki (average) 10000 MC-1 Ki (average)  2250 MC-1 EC₅₀(average; cAMP HBL)  332 MC-1 E_(max) (average; cAMP HBL)  102% 9.66

Assay Result MC-4 Ki (average) 185 MC-1 Ki (average)  3 MC-1 EC₅₀(average; cAMP HBL)  0.18 MC-1 E_(max) (average; cAMP HBL)  97%

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverall such modifications and equivalents. The entire disclosures of allreferences, applications, patents, and publications cited above arehereby incorporated by reference.

We claim:
 1. A cyclic peptide of the formula:

including all enantiomers, stereoisomers or diastereoisomers thereof, ora pharmaceutically acceptable salt of any of the foregoing, wherein: R₄is —(CH₂)_(w)—R₁₄; R₇ is —(CH₂)_(w)—R₁₆; R₉ is—(CH₂)_(x)—C(═O)—NH—(CH₂)_(y)—, —(CH₂)_(x)—NH—C(═O)—(CH₂)_(y)—,—(CH₂)_(x)—C(═O)—(CH₂)_(z)—C(═O)—(CH₂)_(y)—,—(CH₂)_(x)—C(═O)—NH—C(═O)—(CH₂)_(y)—, —(CH₂)_(x)—NH—C(═O)—NH—(CH₂)_(y)—,—(CH₂)_(x)—NH—C(═O)—(CH₂)_(z)—C(═O)—NH—(CH₂)_(y)—, or—(CH₂)_(x)—S—S—(CH₂)_(y)—; R₁₁ is H or a C₁ to C₇ acyl group, whereinthe C₁ to C₁₇ comprises a linear or branched alkyl, cycloalkyl,alkylcycloalkyl, aryl or alkylaryl; R₁₄ is independently in eachinstance —N(R_(19a))(R_(19b)), —NH—(CH₂)_(z)—N(R_(19a))(R_(19b)),—NH—C(═NH)—N(R_(19a))(R_(19b)), —NH—C(═O)—N(R_(19a))(R_(19b)),—O(R_(19a)), —(R_(19a))(R_(19b)), —S(═O)₂(R_(19a)), —C(═O)—O(R_(19a)),

wherein any ring in R₁₄ is optionally substituted with one or more ringsubstituents, and when one or more substituents are present, are thesame or different and independently hydroxyl, halogen, sulfonamide,alkyl, —O-alkyl, aryl, —O-aryl, —C(═O)—OH, or—C(═O)—N(R_(19a))(R_(19b)); R₁₆ is —H, —N(R_(19a))(R_(19b)),—NH—(CH₂)_(z)—N(R_(19a))(R_(19b)), —NH—C(═NH)—N(R_(19a))(R_(19b)),—NH—C(═O)—N(R_(19a))(R_(19b)), —O(R_(19a)), a linear or branched C₁ toC₁₇ alkyl chain, —C(═O)—N(R_(19a))(R_(19b)), —S(═O)₂(R_(19a)),

wherein any ring is optionally substituted with one or more optionalring substituents, and when one or more substituents are present, arethe same or different and independently hydroxyl, halogen, sulfonamide,alkyl, —O-alkyl, aryl, aralkyl, O-aralkyl, or —O-aryl; R₁₈ is —OH,—N(R_(19a))(R_(19b)), —N(R_(19a))(CH₂)_(w) (C₁-C₇)cycloalkyl, or—O—(CH₂)_(w)—(C₁-C₇)cycloalkyl; R_(19a) and R_(19b) are eachindependently H or a C₁ to C₄ linear or branched alkyl chain; R₂₀ isnaphthyl, optionally substituted with one or more substituentsindependently selected from halo, (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl,(C₁-C₁₀)alkoxy, (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro, nitrile,sulfonamide, amino, monosubstituted amino, disubstituted amino, hydroxy,carboxy, and alkoxy-carbonyl; R_(21a), R_(21b) and R_(21c) areindependently in each instance hydrogen, halo, (C₁-C₁₀)alkyl-halo,(C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy, (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro,nitrile, sulfonamide, amino, monosubstituted amino, disubstituted amino,hydroxy, carboxy, or alkoxy-carbonyl; R₂₂ is H or a C₁ to C₉ linear orbranched alkyl, cycloalkyl, alkylcycloalkyl, aryl or alkylaryl; w is ineach instance independently 0 to 5; x is 1 to 5; y is 1 to 5; and z isin each instance independently 1 to
 5. 2. The cyclic peptide of claim 1wherein R₄ is

R₉ is —(CH₂)_(x)—C(═O)—NH—(CH₂)_(y)— where x is 2 and y is 2, or where xis 2 and y is 1; R₇ is

R₁₁ is a C₁ to C₁₇ acyl group, wherein the C₁ to C₁₇ comprises a linearalkyl; R₁₈ is —OH or —NH₂; R_(21a), R_(21b) and R_(21c) are each H; andR₂₂ is a C₁ to C₉ linear or branched alkyl.
 3. The cyclic peptide ofclaim 1 of the formula:


4. The cyclic peptide of claim 1 of the formula:


5. The cyclic peptide of claim 3 of the formula:

wherein R₁₁ is a C₁ to C₁₇ acyl group, wherein the C₁ to C₁₇ comprises alinear alkyl; R₁₈ is —OH or —NH₂; and R₂₂ is a C₁ to C₉ linear orbranched alkyl.
 6. The cyclic peptide of claim 4 of the formula:

wherein R₁₁ is a C₁ to C₁₇ acyl group, wherein the C₁ to C₁₇ comprises alinear alkyl; R₁₈ is —OH or —NH₂; and R₂₂ is a C₁ to C₉ linear orbranched alkyl.
 7. The cyclic peptide of claim 6 of the formula:


8. A cyclic peptide selected from the group consisting of:Ac-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-D-Nal 1-NH₂ (SEQ ID NO:12),Ac-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-Nal 2-NH₂ (SEQ ID NO:13),Ac-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-D-Nal 2-NH₂ (SEQ ID NO:14), andAc-Nle-cyclo(Glu-His-D-Phe-Arg-Dab)-Nal 1-NH₂ (SEQ ID NO:33), or apharmaceutically acceptable salt of any of the foregoing.
 9. Apharmaceutical composition comprising a cyclic peptide orpharmaceutically acceptable salt thereof of claim 1 and apharmaceutically acceptable carrier.
 10. A method for treatment of amelanocortin receptor-mediated disease, indication, condition orsyndrome in a human or non-human mammal, comprising the step ofadministering the pharmaceutical composition of claim
 9. 11. A methodfor treating a condition responsive to changes in melanocortin receptorfunction in a human or non-human mammal, comprising the step ofadministering the pharmaceutical composition of claim
 9. 12. Apharmaceutical composition comprising a cyclic peptide orpharmaceutically acceptable salt thereof of claim 7 and apharmaceutically acceptable carrier.
 13. A method for treatment of amelanocortin receptor-mediated disease, indication, condition orsyndrome in a human or non-human mammal, comprising the step ofadministering the pharmaceutical composition of claim
 12. 14. A methodfor treating a condition responsive to changes in melanocortin receptorfunction in a human or non-human mammal, comprising the step ofadministering the pharmaceutical composition of claim
 12. 15. Apharmaceutical composition comprising a cyclic peptide orpharmaceutically acceptable salt thereof of claim 8 and apharmaceutically acceptable carrier.
 16. A method for treatment of amelanocortin receptor-mediated disease, indication, condition orsyndrome in a human or non-human mammal, comprising the step ofadministering the pharmaceutical composition of claim
 15. 17. A methodfor treating a condition responsive to changes in melanocortin receptorfunction in a human or non-human mammal, comprising the step ofadministering the pharmaceutical composition of claim 15.